Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant

ABSTRACT

The present invention is directed to a polymeric carrier cargo complex, comprising as a cargo at least one nucleic acid molecule and as a preferably non-toxic and non-immunogenic polymeric carrier disulfide-crosslinked cationic components for use as an immunostimulating agent or as an adjuvant, wherein the polymeric carrier cargo complex is administered in combination with at least one second nucleic acid molecule, which encodes a protein or peptide. The inventive polymeric carrier cargo complex administered in combination with the second nucleic acid molecule allows for both efficient transfection of nucleic acids into cells in vivo and in vitro and/or for induction of an innate and/or adaptive immune response, preferably dependent on the nucleic acid to be transported as a cargo and on the second nucleic acid molecule. The present invention also provides pharmaceutical compositions, particularly vaccines, comprising the inventive polymeric carrier cargo complex and the second nucleic acid molecule, as well as the use of the inventive polymeric carrier cargo complex and the second nucleic acid molecule for transfecting a cell, a tissue or an organism, as a medicament, for therapeutic purposes as disclosed herein, and/or as an immunostimulating agent or adjuvant, e.g. for eliciting an immune response for the treatment or prophylaxis of diseases as mentioned herein. Finally, the invention relates to kits containing the inventive polymeric carrier cargo complex and the second nucleic acid molecule, the inventive pharmaceutical composition and/or the inventive vaccine or any of its components in one or more parts of the kit.

This application is a continuation of U.S. application Ser. No.15/300,682, filed Sep. 29, 2016, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2015/000706, filed Apr. 1, 2015, which claims benefit ofInternational Application No. PCT/EP2014/000869, filed Apr. 1, 2014, theentire contents of each of which are hereby incorporated by reference.

The sequence listing that is contained in the file named“CRVCP0157USC1.txt”, which is 97 KB (as measured in Microsoft Windows®)and was created on Jun. 17, 2019, is filed herewith by electronicsubmission and is incorporated by reference herein.

The present invention is directed to a polymeric carrier cargo complex,comprising as a cargo at least one first nucleic acid molecule and as apreferably non-toxic and non-immunogenic polymeric carrierdisulfide-crosslinked cationic components for use as animmunostimulating agent or as an adjuvant, wherein the polymeric carriercargo complex is administered in combination with at least one secondnucleic acid molecule, which encodes a protein or peptide. The inventivepolymeric carrier cargo complex administered in combination with thesecond nucleic acid molecule allows for both efficient transfection ofnucleic acids into cells in vivo and in vitro and/or for induction of aninnate and/or adaptive immune response, preferably dependent on thenucleic acid to be transported as a cargo and on the second nucleic acidmolecule. The present invention also provides pharmaceuticalcompositions, particularly vaccines, comprising the inventive polymericcarrier cargo complex and the second nucleic acid molecule, as well asthe use of the inventive polymeric carrier cargo complex and the secondnucleic acid molecule for transfecting a cell, a tissue or an organism,as a medicament, for therapeutic purposes as disclosed herein, and/or asan immunostimulating agent or adjuvant, e.g. for eliciting an immuneresponse for the treatment or prophylaxis of diseases as mentionedherein. Finally, the invention relates to kits containing the inventivepolymeric carrier cargo complex and the second nucleic acid molecule,the inventive pharmaceutical composition and/or the inventive vaccine orany of its components in one or more parts of the kit.

Many diseases today require administration of adjuvants to provide aninnate immune response and, optionally, to support an adaptive immuneresponse, particularly in the context of vaccinations. Some but notnecessarily all of these diseases additionally or alternatively requireadministration of peptide-, protein-, and nucleic acid-based drugs, e.g.the transfection of nucleic acids into cells or tissues. Theserequirements usually represent different aspects in the treatment ofsuch diseases and are typically difficult to address in one approach. Asa consequence, the prior art usually handles such aspects via separateapproaches.

In the above context, vaccination is generally believed to be one of themost effective and cost-efficient ways to prevent or treat diseases.Nevertheless, several problems in vaccine development have proveddifficult to solve: Vaccines are often inefficient for the very youngand the very old; many vaccines need to be given several times, and theprotection they confer wanes over time, requiring boosteradministrations, and, for some diseases such as HIV, development ofefficient vaccines is urgently needed. As generally accepted, many ofthese vaccines would be enabled or improved if they could elicit astronger and more durable immune response.

Accordingly, the development of new efficient and safe adjuvants forvaccination purposes which support induction and maintenance of anadaptive immune response by initiating or boosting a parallel innateimmune response represents a main challenging problem.

Adjuvants are usually defined as compounds that can increase and/ormodulate the intrinsic immunogenicity of an antigen. To reduce negativeside effects, new vaccines have a more defined composition that oftenleads to lower immunogenicity compared with previous whole-cell orvirus-based vaccines. Adjuvants are therefore required to assist newvaccines to induce potent and persistent immune responses, with theadditional benefit that less antigen and fewer injections are needed.Now it is clear that the adaptive immune response mainly depends on thelevel and specificity of the initial danger signals perceived by innateimmune cells following infection or vaccination (Guy, B. (2007), Nat RevMicrobiol 5(7): 505-17.). In particular for new generation vaccinecandidates, which will increasingly comprise highly purified recombinantproteins and, although very safe, are poorly immunogenic, efficientadjuvants will become increasingly necessary.

Unfortunately, only a few licensed adjuvants are available so far. Mostprominent is Alum, which is known to be safe, but also represents a veryweak adjuvant. Many further adjuvants have been developed, e.g.including the administration of pathogens, CpG-nucleotides, etc. Most ofthese new or “established” adjuvants, however, still do not satisfy theabove requirements, since many new and emerging problems have to beconsidered and solved. These problems inter alia include new andre-emerging infectious diseases, repeated administrations, and threat ofpandemic flu.

Furthermore, the new vaccine targets are usually more difficult todevelop and—due to their specifically tailored immune responses—requiremore potent adjuvants to enable success. Moreover, there are still asignificant number of important pathogens for which we do not even haveeffective vaccines at present. This represents a very challenging futuretarget. To enable vaccine development against such targets, more potentadjuvants will be necessary. Such new adjuvants will need to offeradvantages, including more heterologous antibody responses, coveringpathogen diversity, induction of potent functional antibody responses,ensuring pathogen killing or neutralization and induction of moreeffective T cell responses, for direct and indirect pathogen killing,particularly the induction of cytotoxic T cells which are part of a Th1immune response. In addition, adjuvants may be necessary to achieve morepragmatic effects, including antigen dose reduction and overcomingantigen competition in combination vaccines. Moreover, against thebackground of an aging population, which is increasingly susceptible toinfectious diseases, new adjuvants will be necessary to overcome thenatural deterioration of the immune response with age (O'Hagan, D. T.and E. De Gregorio (2009), Drug Discov Today 14(11-12): 541-51.).

The review of O'Hagan (2009; supra) summarizes some reasons for theurgent need of new effective adjuvants, e.g. the requirement of a lowerantigen dose in vaccines, the necessity to increase the breadth of animmune response and the heterologous activity, to enable complexcombination vaccines, and to overcome antigenic competition, to overcomelimited immune response in some groups of the population, such as theelderly, the young children, and infants, patients with chronic diseasesand the immunocompromised, to increase effector T cell response andantibody titers, to induce protective responses more rapidly and also toextend the duration of response by enhancing memory B and T cellresponses.

Summarizing the above, new efficient and safe immunostimulating agentsor adjuvants are required, which are preferably efficient in inducing aninnate immune response, particularly in inducing the anti-viral cytokineIFN-alpha; which are, preferably, also efficient in supporting anadaptive immune response; safe, i.e. not associated with any long-termeffects; which are well tolerated; which are available via a simplesynthetic pathway; which exhibit low cost storage conditions(particularly feasible lyophilisation); which require simple andinexpensive components; which are biodegradable; which are compatiblewith many different kinds of vaccine antigens; which are capable ofcodelivery of antigen and immune potentiator, etc.

As already explained above, adjuvants or immunostimulating agentsusually act via their capability to induce an innate immune response.The innate immune system forms the dominant system of host defense inmost organisms and comprises barriers such as humoral and chemicalbarriers including, e.g., inflammation, the complement system andcellular barriers. The innate immune system is typically based on asmall number of receptors, called pattern recognition receptors. Theyrecognize conserved molecular patterns that distinguish foreignorganisms, like viruses, bacteria, fungi and parasites, from cells ofthe host. Such pathogen-associated molecular patterns (PAMP) includeviral nucleic acids, components of bacterial and fungal walls, flagellarproteins, and more. The first family of pattern recognition receptors(PAMP receptors) studied in detail was the Toll-like receptor (TLR)family. TLRs are transmembrane proteins which recognize ligands of theextracellular milieu or of the lumen of endosomes. Followingligand-binding they transduce the signal via cytoplasmic adaptorproteins which leads to triggering of a host-defence response andentailing production of antimicrobial peptides, proinflammatorychemokines and cytokines, antiviral cytokines, etc. (see e.g. Meylan,E., J. Tschopp, et al. (2006), Nature 442(7098): 39-44). Furtherrelevant components of the immune system include e.g. the endosomalTLRs, cytoplasmic receptors, Type I interferons and cytoplasmicreceptors. Therefore, the immunostimulating agents or adjuvants aredefined herein preferably as inducers of an innate immune response,which active pattern recognition receptors (PAMP receptors). Hereby, acascade of signals is elicited, which e.g. may result in the release ofcytokines (e.g. IFN-alpha) supporting the innate immune response.Accordingly, it is preferably a feature of an immunostimulating agent oradjuvant to bind to such receptors and activate such PAMP receptors.Ideally, such as an agent or adjuvant additionally supports the adaptiveimmune response by e.g. shifting the immune response such that thepreferred class of Th cells is activated. Depending on the disease ordisorder to be treated a shift to a Th1-based immune response may bepreferred or, in other cases, a shift to a Th2 immune response may bepreferred.

In the prior art there are some promising adjuvant candidates whichfulfil at least some, but not all, of the above defined requiredcharacteristics.

As an example, among the above developed new adjuvants, some nucleicacids, like CpG DNA oligonucleotides or isRNA (immunostimulating RNA),turned out to be promising candidates for new immunostimulating agentsor adjuvants as they allow the therapeutic or prophylactic induction ofan innate immune response. Such nucleic acid based adjuvants usuallyhave to be delivered effectively to the site of action to allowinduction of an effective innate immune response without unnecessaryloss of adjuvant activity and, in some cases, without the necessity toincrease the administered volume above systemically tolerated levels.

One approach to solve this issue may be the transfection of cells whichare part of the innate immune system (e.g. dendritic cells, plasmacytoiddendritic cells (pDCs)) with immunostimulatory nucleic acids, which areligands of PAMP receptors, (e.g. Toll-like receptors (TLRs)), and thusmay lead to immunostimulation by the nucleic acid ligand. Furtherapproaches may be the direct transfection of nucleic acid basedadjuvants. All of these approaches, however, are typically limited byinefficient delivery of the nucleic acid and consequently diminishedadjuvant activity, in particular when administered locally.

However, one main disadvantage of such nucleic acid based adjuvantapproaches until today is their limited ability to cross the plasmamembrane of mammalian cells, resulting in poor cellular access andinadequate therapeutic efficacy. Until today this hurdle represents amajor challenge for nucleic acid transfection based applications, e.g.biomedical developments and accordingly the commercial success of manybiopharmaceuticals (see e.g. Foerg, C. & Merkle, H. P., J Pharm Sci 97,144-62 (2008).

Transfection of nucleic acids or genes into cells or tissues has beeninvestigated up to date in the context of in vitro transfection and inthe context of gene therapeutic approaches. However, no adjuvants areavailable so far which are based on such gene delivery techniques whichare efficient and safe, in particular no licensed adjuvants. This ispresumably due to the complex requirements of adjuvants in general incombination with stability issues to be solved in the case of nucleicacid based adjuvants.

Nevertheless, transfection of nucleic acids or genes into cells ortissues for eliciting an innate and/or adaptive immune response appearsto provide a promising approach to provide new adjuvants.

However, many of these approaches utilize transfection of nucleic acidsor genes into cells or tissues without induction of an innate immuneresponse. There are even some gene therapies, which have to strictlyavoid induction of an innate immune response. Even in the rare cases,where vaccination is carried out to induce an adaptive antigen-specificimmune response using administration of nucleic acids, e.g. in tumourvaccinations using DNA or mRNA encoded antigens, induction of anadaptive immune response is typically carried out as an activeimmunization against the encoded antigen but not as an accompanyingadjuvant therapy and thus requires additional administration of aseparate adjuvant to induce an innate immune response. Even if a seriesof transfection methods are known in the art, transfer or insertion ofnucleic acids or genes into an individual's cells still represents amajor challenge today and is not yet solved satisfactorily. To addressthis complex issue a variety of methods were developed in the lastdecade. These include transfection by calcium phosphate, cationiclipids, cationic polymers, and liposomes. Further methods fortransfection are electroporation and viral transduction.

However, as known to a skilled person, systems for transfer or insertionof nucleic acids or genes have to fulfil several requirements for invivo applications which include efficient nucleic acid delivery into anindividual's cells with high functionality, protection of the nucleicacid against ubiquitously occurring nucleases, release of the nucleicacid in the cell, no safety concerns, feasible manufacturing in acommercially acceptable form amenable to scale-up and storage stabilityunder low cost conditions (e.g feasible lyophilisation). Theserequirements are to be added to the complex requirements of an adjuvantparticularly if it is in the form of a nucleic acid as outlined above.

Some successful strategies for the transfer or insertion of nucleicacids or genes available today rely on the use of viral vectors, such asadenoviruses, adeno-associated viruses, retroviruses, and herpesviruses. Viral vectors are able to mediate gene transfer with highefficiency and the possibility of long-term gene expression. However,the acute immune response (“cytokine storm”), immunogenicity, andinsertion mutagenesis observed in gene therapy clinical trials haveraised serious safety concerns about some commonly used viral vectors.

Another solution to the problem of transfer or insertion of nucleicacids or genes may be found in the use of non-viral vectors. Althoughnon-viral vectors are not as efficient as viral vectors, many non-viralvectors have been developed to provide a safer alternative. Methods ofnon-viral nucleic acid delivery have been explored using physical(carrier-free nucleic acid delivery) and chemical approaches (syntheticvector-based nucleic acid delivery). Physical approaches usually includeneedle injection, electroporation, gene gun, ultrasound, andhydrodynamic delivery, employ a physical force that permeates the cellmembrane and facilitates intracellular gene transfer. The chemicalapproaches typically use synthetic or naturally occurring compounds(e.g. cationic lipids, cationic polymers, lipid-polymer hybrid systems)as carriers to deliver the nucleic acid into the cells. Althoughsignificant progress has been made in the basic science and applicationsof various nonviral nucleic acid delivery systems, the majority ofnon-viral approaches are still much less efficient than viral vectors,especially for in vivo gene delivery (see e.g. Gao, X., Kim, K. & Liu,D., AAPS J 9, E92-104 (2007)).

Such transfection agents as defined above typically have been usedsuccessfully solely in in vitro reactions. For application of nucleicacids in vivo, however, further requirements have to be fulfilled. Forexample, complexes between nucleic acids and transfection agents have tobe stable in physiological salt solutions with respect to aggregation.Furthermore, such complexes typically must not interact with parts ofthe complement system of the host and thus must not be immunogenicitself as the carrier itself shall not induce an adaptive immuneresponse in the individual. Additionally, the complex shall protect thenucleic acid from early extracellular degradation by ubiquitouslyoccurring nucleases.

In the art many transfection reagents are available, especially cationiclipids, which show excellent transfection activity in cell culture.However, most of these transfection reagents do not perform well in thepresence of serum, and only a few are active in vivo. A dramatic changein size, surface charge, and lipid composition occurs when lipoplexesare exposed to the overwhelming amount of negatively charged and oftenamphipathic proteins and polysaccharides that are present in blood,mucus, epithelial lining fluid, or tissue matrix. Once administered invivo, lipoplexes tend to interact with negatively charged bloodcomponents and form large aggregates that could be absorbed onto thesurface of circulating red blood cells, trapped in a thick mucus layer,or embolized in microvasculatures, preventing them from reaching theintended target cells in the distal location. Some even undergodissolution after they are introduced to the blood circulation (see e.g.Gao, X., Kim, K. & Liu, D., AAPS J 9, E92-104 (2007)).

One more promising approach utilizes cationic polymers. Cationicpolymers turned out to be efficient in transfection of nucleic acids, asthey can tightly complex and condense a negatively charged nucleic acid.Thus, a number of cationic polymers have been explored as carriers forin vitro and in vivo gene delivery. These include polyethylenimine(PEI), polyamidoamine and polypropylamine dendrimers, polyallylamine,cationic dextran, chitosan, cationic proteins and cationic peptides.Although most cationic polymers share the function of condensing DNAinto small particles and facilitate cellular uptake via endocytosisthrough charge-charge interaction with anionic sites on cell surfaces,their transfection activity and toxicity differs dramatically.

Only in one approach in the art, the immunostimulatory effect of RNAcomplexed to short cationic peptides was demonstrated by Fotin-Mleczeket al. (WO 2009/030481). These formulations appear to efficiently inducethe cytokine production in immunocompetent cells. UnfortunatelyFotin-Mleczek et al. did not assess the induction of the preferableanti-viral cytokine IFN-α by these complexes. Additionally, thesecomplexes turned out to be unstable during lyophilisation.

In the above context, cationic polymers exhibit better transfectionefficiency with rising molecular weight. However, a rising molecularweight also leads to a rising toxicity of the cationic polymer. In thisabove context, high molecular weight PEI is perhaps the most active andmost studied polymer for transfection of nucleic acids, in particularfor gene delivery purposes. Unfortunately, it exhibits the same drawbackdue to its non-biodegradable nature and toxicity. Furthermore, eventhough polyplexes formed by high molecular weight polymers exhibitimproved stability under physiological conditions, data have indicatedthat such polymers can hinder vector unpacking. To overcome thisnegative impact, Read et al. (see Read, M. L. et al., J Gene Med. 5,232-245 (2003); and Read, M. L. et al., Nucleic Acids Res 33, e86(2005)) developed a new type of synthetic vector based on a linearreducible polycation (RPC) prepared by oxidative polycondensation of thepeptide Cys-Lys₁₀-Cys. This peptide Cys-Lys₁₀-Cys can be cleaved in theintracellular environment to facilitate release of nucleic acids. Inthis context, Read et al. (2003, supra) could show that polyplexesformed by these RPCs are destabilised by reducing conditions enablingefficient release of DNA and mRNA. However, examining the transfectionefficiency in vitro Read et al. (2003, supra) also observed that N/P(nitrogen to phosphor atoms) ratios of 2 were unsatisfying and higherN/P ratios were necessary to improve transfection efficiency.Additionally, Read et al. (2003, supra) observed that chloroquine or thecationic lipid DOTAP was additionally necessary to enhance transfectionefficiency to adequate levels. As a consequence, Read et al. (2005,supra) included histidine residues into the RPCs which have a knownendosomal buffering capacity and showed that such histidine-rich RPCscan be cleaved by the intracellular reducing environment. This approachenabled efficient cytoplasmic delivery of a broad range of nucleicacids, including plasmid DNA, mRNA and siRNA molecules without therequirement for the endosomolytic agent chloroquine.

Unfortunately, neither Read et al. (2003, supra) nor Read et al. (2005,supra) did assess as to whether RPCs can be directly used for in vivoapplications. In their study in 2005, transfections were performed inthe absence of serum to avoid masking the ability of histidine residuesto enhance gene transfer that may have arisen from binding of serumproteins to polyplexes restricting cellular uptake. Preliminaryexperiments, however, indicated that the transfection properties ofhistidine-rich RPC polyplexes can be affected by the presence of serumproteins with a 50% decrease in GFP-positive cells observed in 10% FCS.For in vivo application Read et al. (2005, supra) proposed modificationswith the hydrophilic polymer poly-[N-(2hydroxy-propyl)methacrylamide].Unfortunately, they could not prevent aggregation of polyplexes andbinding of polycationic complexes to serum proteins. Furthermore, strongcationic charged complexes are formed (positive zeta potential) whencomplexing the nucleic acid due to the large excess of cationic polymer,which is characterized by the high N/P ratio. Accordingly, suchcomplexes are only of limited use in vivo due to their strong tendencyof salt induced aggregation and interactions with serum contents.Additionally, these (positively charged) complexes may excite complementactivation, when used for purposes of gene therapy. It has also turnedout that these positively charged RPC based complexes showed poortranslation of the nucleic acid cargo subsequent to local administrationinto the dermis.

In an approach similar to Read et al. McKenzie et al. (McKenzie, D. L.,K. Y. Kwok, et al. (2000), J Biol Chem 275(14): 9970-7. and McKenzie, D.L., E. Smiley, et al. (2000), Bioconjug Chem 11(6): 901-9) developedcross-linking peptides as gene delivery agents by inserting multiplecysteines into short synthetic peptides. In their studies they examinedthe optimal complex formation with DNA and as a result they could showthat an N/P ratio of at least 2 is necessary for fully formed peptideDNA condensates. Therefore only positively charged complexes appeared toshow optimal DNA condensation. In contrast to these data they proposedthe development of negatively charged complexes for in vivo genedelivery, since it was shown in previous studies that intravenousapplication of electropositive DNA condensates leads to rapidopsonisation and nonspecific biodistribution to lung and liver (Collard,W. T., Evers, D. L., McKenzie, D. L., and Rice, K. G. (2000), Carbohydr.Res. 323, 176-184). Therefore McKenzie et al. (2000; supra) proposed thederivatization of the carriers with polyethylene glycol and targetingligands. To be noted, the approach of McKenzie et al. (2000, supra) isadditionally subject of a patent (U.S. Pat. No. 6,770,740 B1), whichparticularly discloses the transfection of coding nucleic acids,antisense nucleic acids and ribozymes.

Thus, in vivo application of nucleic acids appears to be still one ofthe most challenging problems because plasma proteins with anioniccharges may non-specifically bind to positively charged complexes andrapidly remove them e.g. via the reticulo-endothelial system.Opsonization and activation of the complement system by cationiccomplexes are additional physiological phenomena that can participate inlowering the efficacy of in vivo administered cationic complexes. Thisparticularly applies to administration of nucleic acid-based drugs, e.g.the transfection of nucleic acids into cells or tissues, particularly ifthe expression of an encoded protein or peptide or transcription of anRNA of the transfected nucleic acid is intended. In particular, therecontinues to be a great need for a system that allows administration ofnucleic acid-based drugs, particularly nucleic acid-based drugscomprising an adjuvant function, by a method, which warrants a highlevel of safety and efficacy and which can readily be applied in avariety of situations and without specific training.

Summarizing the above, the prior art does not provide feasible means ormethods, which, on the one hand, allow to establish efficient and safeadjuvants for vaccination purposes, and which, on the other hand, arefurthermore suited for in vivo delivery of nucleic acids, in particularfor compacting and stabilizing a nucleic acid for the purposes ofnucleic acid transfection in vivo without exhibiting the negative sideeffects as discussed above. More precisely, no means or methods areknown in the prior art in the above context, which are, on the one hand,stable enough to carry a nucleic acid cargo to the target before theyare metabolically cleaved, and which, on the other hand, can be clearedfrom the tissue before they can accumulate and reach toxic levels. Inaddition no means or method is known, which, additional to the aboverequirements, induces a desirable pattern of cytokines, particularly theanti viral cytokine IFN-α.

Accordingly, it is the object of the present invention to provide suchmeans or methods, which address these problems.

The object underlying the present invention is solved by the subjectmatter of the present invention, preferably by the subject matter of theattached claims.

According to a first aspect, the object underlying the present inventionis solved by a polymeric carrier cargo complex, comprising or consistingof

-   -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components and    -   b) as a cargo at least one first nucleic acid molecule,        preferably for use as a medicament, more preferably for use as        an immunostimulating agent or adjuvant, e.g. in the treatment of        a disease as defined herein, wherein the polymeric carrier cargo        complex is administered in combination with at least one second        nucleic acid molecule, which encodes a protein or a peptide, and        wherein the polymeric carrier cargo complex and the second        nucleic acid molecule are administered intramuscularly.

In a preferred embodiment, the invention relates to a polymeric carriercargo complex, comprising:

-   -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components, and    -   b) as a cargo at least one first nucleic acid molecule, for use        as an immunostimulating agent or as an adjuvant,        wherein the polymeric carrier cargo complex is administered in        combination with at least one second nucleic acid molecule        encoding a protein or a peptide, and wherein the polymeric        carrier cargo complex and the second nucleic acid molecule are        administered intramuscularly.

Alternatively, the problem is solved by a polymeric carrier cargocomplex, comprising:

-   -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components, and    -   b) as a cargo at least one first nucleic acid molecule,        preferably for use as a medicament, more preferably for use as        an immunostimulating agent or as an adjuvant, e.g. in the        treatment of a disease as defined herein,        wherein the polymeric carrier cargo complex is administered in        combination with at least one second nucleic acid molecule        encoding a protein or a peptide, and        wherein the second nucleic acid molecule is an RNA molecule,        preferably an mRNA molecule.

In a preferred embodiment, the invention relates to a polymeric carriercargo complex, comprising:

-   -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components, and    -   b) as a cargo at least one first nucleic acid molecule,        for use as an immunostimulating agent or as an adjuvant, wherein        the polymeric carrier cargo complex is administered in        combination with at least one second nucleic acid molecule        encoding a protein or a peptide, wherein the second nucleic acid        molecule is an RNA molecule, preferably an mRNA molecule.

As used herein, the term “first nucleic acid molecule” refers to anucleic molecule, which is used as a cargo in the polymeric carriercargo complex and is thus associated with the polymeric carrier. Theterm “second nucleic acid molecule”, as used herein, typically refers toa nucleic acid, which is not part of the polymeric carrier cargo complexand which encodes a peptide or protein.

The term “immunostimulating agent” is typically understood not toinclude agents as e.g.

antigens (of whatever chemical structure), which elicit anadaptive/cytotoxic immune response, e.g. a “humoral” or “cellular”immune response, in other words elicit immune responses (and conferimmunity by themselves) which are characterized by a specific responseto structural properties of an antigen recognized to be foreign byimmune competent cells. Rather “immunostimulating agent” is typicallyunderstood to mean agents/compounds/complexes which do not trigger anyadaptive immune response by themselves, but which may exclusivelyenhance such an adaptive immune response in an unspecific way, by e.g.activating “PAMP” receptors and thereby triggering the release ofcytokines which support the actual adaptive immune response.Accordingly, any immunostimulation by agents (e.g. antigens) which evokean adaptive immune response by themselves (conferring immunity bythemselves directly or indirectly) is typically disclaimed by the phrase“immunostimulating agent”.

The term “adjuvant” is also understood not to comprise agents whichconfer immunity by themselves. Accordingly, adjuvants do not bythemselves confer immunity, but assist the immune system in various waysto enhance the antigen-specific immune response by e.g. promotingpresentation of an antigen to the immune system. Hereby, an adjuvant maypreferably e.g. modulate the antigen-specific immune response by e.g.shifting the dominating Th2-based antigen specific response to a moreTh1-based antigen specific response or vice versa. Accordingly, theterms “immunostimulating agent” and “adjuvant” in the context of thepresent invention are typically understood to mean agents, compounds orcomplexes which do not confer immunity by themselves, but exclusivelysupport the immune response in an unspecific way (in contrast to anantigen-specific immune response) by effects, which modulate theantigen-specific (adaptive cellular and/or humoral immune response) byunspecific measures, e.g. cytokine expression/secretion, improvedantigen presentation, shifting the nature of the arms of the immuneresponse etc. Accordingly, any agents evoking by themselves immunity aretypically disclaimed by the terms “adjuvant” or “immunostimulatingagent”.

The use of the polymeric carrier cargo complex in combination with asecond nucleic acid molecule, preferably an RNA, allows provision of amore efficient and/or safer medicament. Advantageously, the polymericcarrier cargo complex is suited for in vivo delivery of nucleic acids,in particular for compacting and stabilizing a nucleic acid for thepurposes of nucleic acid transfection, such as exhibiting one or morereduced negative side effects of high-molecular weight polymers asdiscussed above, such as poor biodegradability or high toxicity,agglomeration, low transfection activity in vivo, etc. The polymericcarrier cargo complex also provides for improved nucleic acid transferin vivo, particularly via intradermal or intramuscular routes, includingserum stability, salt stability, efficiency of uptake, reducedcomplement activation, nucleic acid release, etc. Such a polymericcarrier cargo complex furthermore may support induction and maintenanceof an adaptive immune response by initiating or boosting a parallelinnate immune response. It has been found that an improved adaptiveimmune response can further be obtained, in particular when thepolymeric carrier cargo complex is administered in combination with asecond nucleic acid molecule, preferably an RNA, encoding a protein orpeptide, or when the polymeric carrier cargo complex is co-formulated ina pharmaceutical composition with a second nucleic acid molecule,preferably an RNA, encoding a protein or peptide, preferably anantigenic peptide or protein. It has proven as particularly beneficialin this respect to administer the pharmaceutical composition as definedherein or the polymeric carrier cargo complex in combination with thesecond nucleic acid molecule as defined herein via an intramuscularroute. Additionally, the polymeric carrier cargo complex may exhibitimproved storage stability, particularly during lyophilisation.

In particular, the polymeric carrier cargo complex as defined aboveenhances the immune response against a protein or peptide, which isencoded by a second nucleic acid molecule, preferably an RNA, morepreferably an mRNA, that is administered in combination with thepolymeric carrier cargo complex, preferably via an intramuscular routeof administration.

The polymeric carrier cargo complex and/or the second nucleic acidmolecule encoding a peptide or protein are preferably provided togetherwith a pharmaceutically acceptable carrier and/or vehicle. In thecontext of the present invention, a pharmaceutically acceptable carriertypically includes the liquid or non-liquid material, which is mixedwith the polymeric carrier cargo complex and/or the second nucleic acidmolecule. If the polymeric carrier cargo complex and/or the secondnucleic acid molecule are provided in liquid form, the carrier willtypically be pyrogen-free water; isotonic saline or buffered aqueoussolutions, e.g phosphate, citrate etc. buffered solutions. Ringer orRinger-Lactate solution is particularly preferred as a liquid basis.

The phrase “administered in combination” as used herein refers to asituation, where the polymeric carrier cargo complex is administered toa subject before, concomittantly or after the administration of thesecond nucleic acid molecule encoding a protein or peptide to the samesubject. Preferably, the time interval between the administration of thepolymeric carrier cargo complex and the at least one second nucleic acidmolecule, preferably an RNA, encoding a protein or peptide is less thanabout 48 hours, more preferably less than about 24 hours, 12 hours, 6hours, 4 hours, 2 hours, 1 hour, most preferably less than about 30minutes, 15 minutes or 5 minutes. In a particularly preferredembodiment, the phrase “administered in combination” refers toconcomitant administration of the polymeric carrier cargo complex andthe at least one second nucleic acid molecule, i.e. the simultaneousadministration of both components or the administration of bothcomponents within a time frame that typically comprises less than 5minutes. The phrase “administered in combination” does not only refer toa situation, where the pharmaceutical carrier cargo complex is inphysical contact with the at least one second nucleic acid molecule orformulated together with said second nucleic acid molecule. The phrase“administered in combination” as used herein comprises also the separateadministration of the polymeric carrier cargo complex and the secondnucleic acid molecule (e.g. by two separate intramuscular injections),as long as the time interval between the two injections does not exceedthe interval as defined above. Alternatively, the polymeric carriercargo complex and the second nucleic acid molecule may be administeredin combination by mixing the polymeric carrier cargo complex and thesecond nucleic acid molecule prior to administration and administeringthe mixture to a subject. When the polymeric carrier cargo complex isformulated together with the second nucleic acid molecule or when apharmaceutical composition as defined herein is used, the polymericcarrier cargo complex and the second nucleic acid molecule may further,independently from each other, administered in combination via any ofthe administration routes as described herein.

According to a preferred embodiment, the second nucleic acid molecule,which is administered in combination with the polymeric carrier cargocomplex, is not comprised in the polymeric carrier cargo complex. Morepreferably, the second nucleic acid molecule is administered incombination with the polymeric carrier cargo complex as defined herein,without physically being a part or component of the polymeric carriercargo complex. In particular, the second nucleic acid molecule ispreferably not bound (e.g. covalently) to the polymeric carrier cargocomplex. Further preferably, the at least one first nucleic acidmolecule of the inventive polymeric carrier cargo complex and the atleast one second nucleic acid molecule, which is administered togetherwith the polymeric carrier cargo complex, are not complexed by the samepolymeric carrier.

In a further preferred embodiment, the present invention provides apolymeric carrier cargo complex for use as an immunostimulating agent oran adjuvant, wherein the polymeric carrier cargo complex is administeredin combination with at least one second nucleic acid molecule encoding aprotein or a peptide, wherein the polymeric carrier cargo complex andthe second nucleic acid molecule are administered intramuscularly andwherein the polymeric carrier cargo complex and the second nucleic acidmolecule are not administered together with a protein or peptide antigenselected from the group consisting of an antigen from a pathogenassociated with infectious disease, an antigen associated with allergyor allergic disease, an antigen associated with autoimmune disease, anantigen associated with a cancer or tumour disease, or a fragment,variant and/or derivative of said protein or peptide antigen. Morepreferably, the present invention provides a polymeric carrier cargocomplex for use as an immunostimulating agent or an adjuvant, whereinthe polymeric carrier cargo complex is administered in combination withat least one second nucleic acid molecule encoding a protein or apeptide, wherein the polymeric carrier cargo complex and the secondnucleic acid molecule are administered intramuscularly and wherein thepolymeric carrier cargo complex and the second nucleic acid molecule arenot administered together with a protein or peptide antigen.

The inventive polymeric carrier cargo complex as defined above comprisesas one component a polymeric carrier formed by disulfide-crosslinkedcationic components. The term “cationic component” typically refers to acharged molecule, which is positively charged (cation) at a pH value ofabout 1 to 9, preferably of a pH value of or below 9, of or below 8, ofor below 7, most preferably at physiological pH values, e.g. about 7.3to 7.4. Accordingly, a cationic peptide, protein or polymer according tothe present invention is positively charged under physiologicalconditions, particularly under physiological pH value conditions of thecell in vivo. The term “cationic” may also refer to “oligocationic” or“polycationic” components. In the context of the present invention, theterm “oligocationic” further refers to a compound, which preferablycarries from two to five positive charges, i.e. which comprises from twoto five cations, at a pH value of about 1 to 9, preferably of a pH valueof or below 9, of or below 8, of or below 7, most preferably atphysiological pH values, e.g. about 7.3 to 7.4. In this context, theterm “polycationic” typically refers to a compound carrying at least sixpositive charges, i.e. comprising at least six cations, at a pH value ofabout 1 to 9, preferably of a pH value of or below 9, of or below 8, ofor below 7, most preferably at physiological pH values, e.g. about 7.3to 7.4.

Advantageously, in a cationic peptide or protein as used hereinpreferably at least 20% of the amino acid residues of said peptide orprotein, more preferably at least 30% of the amino acid residues of saidpeptide or protein, even more preferably at least 40% of the amino acidresidues of said peptide or protein, most preferably at least 50% of theamino acid residues of said protein or peptide are positively charged.

In this context the cationic components, which form the basis for thepolymeric carrier of the inventive polymeric carrier cargo complex bydisulfide-crosslinkage, are typically selected from any suitablecationic or polycationic peptide, protein or polymer suitable for thispurpose, particular any cationic or polycationic peptide, protein orpolymer capable to complex a nucleic acid as defined according to thepresent invention, and thereby preferably condensing the nucleic acid.The cationic or polycationic peptide, protein or polymer, is preferablya linear molecule, however, branched cationic or polycationic peptides,proteins or polymers may also be used.

Each cationic or polycationic protein, peptide or polymer of thepolymeric carrier contains at least one —SH moiety, most preferably atleast one cysteine residue or any further chemical group exhibiting an—SH moiety, capable to form a disulfide linkage upon condensation withat least one further cationic or polycationic protein, peptide orpolymer as cationic component of the polymeric carrier as mentionedherein.

Each cationic or polycationic protein, peptide or polymer or any furthercomponent of the polymeric carrier is preferably linked to itsneighbouring component(s) (cationic proteins, peptides, polymers orother components) via disulfide-crosslinking. Preferably, thedisulfide-crosslinking is a reversible disulfide bond (—S—S—) between atleast one cationic or polycationic protein, peptide or polymer and atleast one further cationic or polycationic protein, peptide or polymeror other component of the polymeric carrier. The disulfide-crosslinkingis typically formed by condensation of —SH-moieties of the components ofthe polymeric carrier particularly of the cationic components. Such an—SH-moiety may be part of the structure of the cationic or polycationicprotein, peptide or polymer or any further component of the polymericcarrier prior to disulfide-crosslinking or may be added prior todisulfide-crosslinking by a modification as defined below. In thiscontext, the sulphurs adjacent to one component of the polymericcarrier, necessary for providing a disulfide bond, may be provided bythe component itself, e.g. by a —SH moiety as defined herein or may beprovided by modifying the component accordingly to exhibit a —SH moiety.These —SH-moieties are typically provided by each of the component, e.g.via a cysteine or any further (modified) amino acid or compound of thecomponent, which carries a —SH moiety. In the case that the cationiccomponent or any further component of the polymeric carrier is a peptideor protein it is preferred that the —SH moiety is provided by at leastone cysteine residue. Alternatively, the component of the polymericcarrier may be modified accordingly with a —SH moiety, preferably via achemical reaction with a compound carrying a —SH moiety, such that eachof the components of the polymeric carrier carries at least one such —SHmoiety. Such a compound carrying a —SH moiety may be e.g. an(additional) cysteine or any further (modified) amino acid or compoundof the component of the polymeric carrier, which carries a —SH moiety.Such a compound may also be any non-amino compound or moiety, whichcontains or allows to introduce a —SH moiety into the component asdefined herein. Such non-amino compounds may be attached to thecomponent of the polymeric carrier according to the present inventionvia chemical reactions or binding of compounds, e.g. by binding of a3-thio propionic acid or 2-iminothiolane (Traut's reagent), by amideformation (e.g. carboxylic acids, sulphonic acids, amines, etc.), byMichael addition (e.g maleinimide moieties, α,β unsatured carbonyls,etc.), by click chemistry (e.g. azides or alkines), by alkene/alkinemethatesis (e.g. alkenes or alkines), imine or hydrozone formation(aldehydes or ketons, hydrazins, hydroxylamins, amines), complexationreactions (avidin, biotin, protein G) or components which allowS_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols,amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphoniumsalts) or other chemical moieties which can be utilized in theattachment of further components. In some cases the —SH moiety may bemasked by protecting groups during chemical attachment to the component.Such protecting groups are known in the art and may be removed afterchemical coupling. In each case, the —SH moiety, e.g. of a cysteine orof any further (modified) amino acid or compound, may be present at theterminal ends or internally at any position of the component of thepolymeric carrier. As defined herein, each of the components of thepolymeric carrier typically exhibits at least one —SH-moiety, but mayalso contain two, three, four, five, or even more —SH-moieties.Additionally to binding of cationic components a —SH moiety may be usedto attach further components of the polymeric carrier as defined herein,particularly an amino acid component, e.g. antigen epitopes, antigens,antibodies, cell penetrating peptides (e.g. TAT), ligands, etc.

As defined above, the polymeric carrier of the inventive polymericcarrier cargo molecule is formed by disulfide-crosslinked cationic (orpolycationic) components.

According to one first alternative, at least one cationic (orpolycationic) component of the polymeric carrier may be selected fromcationic or polycationic peptides or proteins. Such cationic orpolycationic peptides or proteins preferably exhibit a length of about 3to 100 amino acids, preferably a length of about 3 to 50 amino acids,more preferably a length of about 3 to 25 amino acids, e.g. a length ofabout 3 to 10; 5 to 20; 5 to 15; 8 to 15, 16 or 17; 10 to 15, 16, 17,18, 19, or 20; or 15 to 25 amino acids. Alternatively or additionally,such cationic or polycationic peptides or proteins may exhibit amolecular weight of about 0.1 kDa to about 100 kDa, including amolecular weight of about 0.5 kDa to about 100 kDa, preferably of about10 kDa to about 50 kDa, even more preferably of about 10 kDa to about 30kDa.

In the specific case that the cationic component of the polymericcarrier comprises a cationic or polycationic peptide or protein, thecationic properties of the cationic or polycationic peptide or proteinor of the entire polymeric carrier, if the polymeric carrier is entirelycomposed of cationic or polycationic peptides or proteins, may bedetermined upon its content of cationic amino acids. Preferably, thecontent of cationic amino acids in the cationic or polycationic peptideor protein and/or the polymeric carrier is at least 10%, 20%, or 30%,preferably at least 40%, more preferably at least 50%, 60% or 70%, butalso preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%,more preferably in the range of about 15% to 75%, even more preferablyin the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a rangeformed by any two of the afore mentioned values, provided, that thecontent of all amino acids, e.g. cationic, lipophilic, hydrophilic,aromatic and further amino acids, in the cationic or polycationicpeptide or protein, or in the entire polymeric carrier, if the polymericcarrier is entirely composed of cationic or polycationic peptides orproteins, is 100%.

In this context, cationic amino acids are preferably the naturallyoccurring amino acids Arg (Arginine), Lys (Lysine), His (Histidine), andOrn (Ornithin). However, in a broader sense any non-natural amino acidcarrying a cationic charge on its side chain may also be envisaged tocarry out the invention. Preferably, however, are those cationic aminoacids, the side chains of which are positively charged underphysiological pH conditions. In a more preferred embodiment, these aminoacids are Arg, Lys, and Orn.

Preferably, such cationic or polycationic peptides or proteins of thepolymeric carrier, which comprise or are additionally modified tocomprise at least one —SH moeity, are selected from, without beingrestricted thereto, cationic peptides or proteins such as protamine,nucleoline, spermine or spermidine, oligo- or poly-L-lysine (PLL), basicpolypeptides, oligo or poly-arginine, cell penetrating peptides (CPPs),chimeric CPPs, such as Transportan, or MPG peptides, HIV-bindingpeptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, members of thepenetratin family, e.g. Penetratin, Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIs1, etc.,antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1),pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Loligomere, FGF,Lactoferrin, histones, VP22 derived or analog peptides, HSV, VP22(Herpes simplex), MAP, KALA or protein transduction domains (PTDs),PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.

Alternatively or additionally, such cationic or polycationic peptides orproteins of the polymeric carrier, which comprise or are additionallymodified to comprise at least one —SH moeity, are selected from, withoutbeing restricted thereto, following cationic peptides having thefollowing sum formula (I):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};

wherein l+m+n+o+x=3-100, and l, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may bepositioned at any place of the peptide. In this context cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred. Even more preferred peptides of this formula areoligoarginines such as e.g. Arg₇, Arg₈, Arg₉, Arg₁₂, His₃Arg₉, Arg₉His₃,His₃Arg₉His₃, His₆Arg₉His₆, His₃Arg₄His₃, His₆Arg₄His₆,TyrSer₂Arg₉Ser₂Tyr, (ArgLysHis)₄, Tyr(ArgLysHis)₂Arg, etc.

According to a particular preferred embodiment, such cationic orpolycationic peptides or proteins of the polymeric carrier having theempirical sum formula (I) as shown above, may, without being restrictedthereto, comprise at least one of the following subgroup of formulae:

Arg₇, Arg₈, Arg₉, Arg₁₀, Arg₁₁, Arg₁₂, Arg₁₃, Arg₁₄, Arg₁₅₋₃₀;Lys₇, Lys₈, Lys₉, Lys₁₀, Lys₁₁, Lys₁₂, Lys₁₃, Lys₁₄, Lys₁₅₋₃₀;His₇, His₈, His₉, His₁₀, His₁₁, His₁₂, His₁₃, His₁₄, His₁₅₋₃₀;Orn₇, Orn₈, Orn₉, Orn₁₀, Orn₁₁, Orn₁₂, Orn₁₃, Orn₁₄, Orn₁₅₋₃₀;

According to a further particularly preferred embodiment, cationic orpolycationic peptides or proteins of the polymeric carrier, having theempirical sum formula (I) as shown above and which comprise or areadditionally modified to comprise at least one —SH moeity, may bepreferably selected from, without being restricted thereto, at least oneof the following subgroup of formulae. The following formulae (as withempirical formula (I)) do not specify any amino acid order, but areintended to reflect empirical formulae by exclusively specifying the(number of) amino acids as components of the respective peptide.Accordingly, as an example, empirical formula Arg₍₇₋₂₉₎Lys₁ is intendedto mean that peptides falling under this formula contain 7 to 19 Argresidues and 1 Lys residue of whatsoever order. If the peptides contain7 Arg residues and 1 Lys residue, all variants having 7 Arg residues and1 Lys residue are encompassed. The Lys residue may therefore bepositioned anywhere in the e.g. 8 amino acid long sequence composed of 7Arg and 1 Lys residues. The subgroup preferably comprises:

Arg₍₄₋₂₉₎Lys₁, Arg₍₄₋₂₉₎His₁, Arg₍₄₋₂₉Orn₁,Lys₍₄₋₂₉₎His₁, Lys₍₄₋₂₉₎Orn₁, His₍₄₋₂₉₎Orn₁,Arg₍₃₋₂₈₎Lys₂, Arg₍₃₋₂₈₎His₂, Arg₍₃₋₂₈₎Orn₂,Lys₍₃₋₂₈₎HiS₂, Lys₍₃₋₂₈₎Orn₂, His₍₃₋₂₈₎Orn₂,Arg₍₂₋₂₇₎Lys₃, Arg₍₂₋₂₇₎His₃, Arg₍₂₋₂₇₎Orn₃,Lys₍₂₋₂₇₎His₃, Lys₍₂₋₂₇₎Orn₃, His₍₂₋₂₇₎Orn₃,Arg₍₁₋₂₆₎Lys₄, Arg₍₁₋₂₆₎His₄, Arg₍₁₋₂₆₎Orn₄,Lys₍₁₋₂₆₎His₄, Lys₍₁₋₂₆₎Orn₄, His₍₁₋₂₆₎Orn₄,Arg₍₃₋₂₈₎Lys₁His₁, Arg₍₃₋₂₈₎Lys₁Orn₁,Arg₍₃₋₂₈₎His₁Orn₁, Arg₁Lys₍₃₋₂₈₎His₁,Arg₁Lys₍₃₋₂₈₎Orn₁, Lys₍₃₋₂₈₎His₁Orn₁,Arg₁Lys₁His₍₃₋₂₈₎, Arg₁His₍₃₋₂₈₎Orn₁, Lys₁His₍₃₋₂₈₎Orn₁;Arg₍₂₋₂₇₎Lys₂His₁, Arg₍₂₋₂₇₎Lys₁His₂,Arg₍₂₋₂₇₎Lys₂Orn₁, Arg₍₂₋₂₇₎Lys₁Orn₂,Arg₍₂₋₂₇₎His₂Orn₁, Arg₍₂₋₂₇₎His₁Orn₂,Arg₂Lys₍₂₋₂₇₎His₁, Arg₁Lys₍₂₋₂₇₎His₂,Arg₂Lys₍₂₋₂₇₎Orn₁, Arg₁Lys₍₂₋₂₇₎Orn₂,Lys₍₂₋₂₇₎His₂Orn₁, Lys₍₂₋₂₇₎His₁Orn₂,Arg₂Lys₁His₍₂₋₂₇₎, Arg₁Lys₂His₍₂₋₂₇₎,Arg₂His₍₂₋₂₇₎Orn₁, Arg₁His₍₂₋₂₇₎Orn₂,Lys₂His₍₂₋₂₇₎Orn₁, Lys₁His₍₂₋₂₇₎Orn₂;Arg₍₁₋₂₆₎Lys₃His₁, Arg₍₁₋₂₆₎Lys₂His₂,Arg₍₁₋₂₆₎Lys₁His₃, Arg₍₁₋₂₆₎Lys₃Orn₁,Arg₍₁₋₂₆₎Lys₂Orn₂, Arg₍₁₋₂₆₎Lys₁Orn₃,Arg₍₁₋₂₆₎His₃Orn₁, Arg₍₁₋₂₆₎His₂Orn₂,Arg₍₁₋₂₆₎His₁Orn₃, Arg₃Lys₍₁₋₂₆₎His₁,Arg₂Lys₍₁₋₂₆₎His₂, Arg₁Lys₍₁₋₂₆₎His₃,Arg₃Lys₍₁₋₂₆₎Orn₁, Arg₂Lys₍₁₋₂₆₎Orn₂,Arg₁Lys₍₁₋₂₆₎Orn₃, Lys₍₁₋₂₆₎His₃Orn₁,Lys₍₁₋₂₆₎His₂Orn₂, Lys₍₁₋₂₆₎His₁Orn₃,Arg₃Lys₁His₍₁₋₂₆₎, Arg₂Lys₂His₍₁₋₂₆₎,Arg₁Lys₃His₍₁₋₂₆₎, Arg₃His₍₁₋₂₆₎Orn₁,Arg₂His₍₁₋₂₆₎Orn₂, Arg₁His₍₁₋₂₆₎Orn₃,Lys₃His₍₁₋₂₆₎Orn₁, Lys₂His₍₁₋₂₆₎Orn₂, Lys₁His₍₁₋₂₆₎Orn₃;Arg₍₂₋₂₇₎Lys₁His₁Orn₁, Arg₁Lys₍₂₋₂₇₎ His₁Orn₁, Arg₁Lys₁His₍₂₋₂₇₎Orn₁,Arg₁Lys₁His₁Orn₍₂₋₂₇₎; Arg₍₁₋₂₆₎Lys₂His₁Orn₁, Arg₍₁₋₂₆₎Lys₁His₂Orn₁,Arg₍₁₋₂₆₎Lys₁His₁Orn₂, Arg₂Lys₍₁₋₂₆₎His₁Orn₁,Arg₁Lys₍₁₋₂₆₎His₂Orn₁, Arg₁Lys₍₁₋₂₆₎His₁Orn₂,Arg₂Lys₁His₍₁₋₂₆₎Orn₁, Arg₁Lys₂His₍₁₋₂₆₎Orn₁,Arg₁Lys₁His_((l-26))Orn₂, Arg₂Lys₁His₁Orn_((l-26)),Arg₁Lys₂His₁Orn₍₁₋₂₆₎, Arg₁Lys₁His₂Orn₍₁₋₂₆₎;

According to a further particular preferred embodiment, cationic orpolycationic peptides or proteins of the polymeric carrier, having theempirical sum formula (I) as shown above and which comprise or areadditionally modified to comprise at least one —SH moeity, may be,without being restricted thereto, selected from the subgroup consistingof generic formulas Arg₇ (also termed as R₇), Arg₉ (also termed R₉),Arg₁₂ (also termed as R₁₂).

According to a one further particular preferred embodiment, the cationicor polycationic peptide or protein of the polymeric carrier, whendefined according to formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (I)) asshown above and which comprise or are additionally modified to compriseat least one —SH moeity, may be, without being restricted thereto,selected from subformula (Ia):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)}  formula(Ia)

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80 and 81-90, provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide.

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (I)) as shownabove, comprises or has been modified with at least one cysteine as —SHmoiety in the above meaning such that the cationic or polycationicpeptide as cationic component carries at least one cysteine, which iscapable to form a disulfide bond with other components of the polymericcarrier.

According to another particular preferred embodiment, the cationic orpolycationic peptide or protein of the polymeric carrier, when definedaccording to formula {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}(formula (I)) as shown above, may be, without being restricted thereto,selected from subformula (Ib):

Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂  (formula(Ib))

wherein empirical formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (I)) is asdefined herein and forms a core of an amino acid sequence according to(semiempirical) formula (I) and wherein Cys₁ and Cys₂ are Cysteinesproximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x). Exemplary examplesmay comprise any of the above sequences flanked by two Cys and followingsequences:

Cys(Arg₇)Cys, Cys(Arg₈)Cys, Cys(Arg₉)Cys,Cys(Arg₁₀)Cys, Cys(Arg₁₁)Cys, Cys(Arg₁₂)Cys,Cys(Arg₁₃)Cys, Cys(Arg₁₄)Cys, Cys(Arg₁₅)Cys,Cys(Arg₁₆)Cys, Cys(Arg₁₇)Cys, Cys(Arg₁₈)Cys,Cys(Arg₁₉)Cys, Cys(Arg₂₀)Cys (SEQ ID NOs: 1-14): CysArg₇Cys(SEQ ID NO. 1) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₈Cys(SEQ ID NO. 2) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₉Cys:(SEQ ID NO. 3) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₀Cys(SEQ ID NO. 4) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-CysCysArg₁₁Cys (SEQ ID NO. 5)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Cys CysArg₁₂Cys:(SEQ ID NO. 6) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-CysCysArg₁₃Cys: (SEQ ID NO. 7)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-CysCysArg₁₄Cys: (SEQ ID NO. 8)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-CysCysArg₁₅Cys: (SEQ ID NO. 9)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Arg-CysCysArg₁₆Cys: (SEQ ID NO. 10)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Arg-Arg-CysCysArg₁₇Cys: (SEQ ID NO. 11)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₈Cys: (SEQ ID NO. 12)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₉Cys: (SEQ ID NO. 13)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₂₀Cys: (SEQ ID NO. 14)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg Cys

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (I)) as shownabove, has been modified with at least two cysteines as —SH moieties inthe above meaning such that the cationic or polycationic peptide of theinventive polymeric carrier cargo complex as cationic component carriesat least two (terminal) cysteines, which are capable to form a disulfidebond with other components of the polymeric carrier.

In a preferred embodiment, the polymeric carrier cargo complex comprisesa carrier, which comprises or consists of the peptide CysArg₁₂Cys (SEQID NO: 6). Therein, the peptide having the sequence according to SEQ IDNO: 6 is preferably further modified by an amino acid component (AA) asdefined herein.

According to a second alternative, at least one cationic (orpolycationic) component of the polymeric carrier may be selected frome.g. any (non-peptidic) cationic or polycationic polymer suitable inthis context, provided that this (non-peptidic) cationic or polycationicpolymer exhibits or is modified to exhibit at least one —SH-moiety,which provide for a disulfide bond linking the cationic or polycationicpolymer with another component of the polymeric carrier as definedherein. Thus, likewise as defined herein, the polymeric carrier maycomprise the same or different cationic or polycationic polymers.

In the specific case that the cationic component of the polymericcarrier comprises a (non-peptidic) cationic or polycationic polymer thecationic properties of the (non-peptidic) cationic or polycationicpolymer may be determined upon its content of cationic charges whencompared to the overall charges of the components of the cationicpolymer. Preferably, the content of cationic charges in the cationicpolymer at a (physiological) pH as defined herein is at least 10%, 20%,or 30%, preferably at least 40%, more preferably at least 50%, 60% or70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%,99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to90%, more preferably in the range of about 30% to 100%, even preferablyin the range of about 50% to 100%, e.g. 50, 60, 70, 80%, 90% or 100%, orin a range formed by any two of the afore mentioned values, provided,that the content of all charges, e.g. positive and negative charges at a(physiological) pH as defined herein, in the entire cationic polymer is100%.

Preferably, the (non-peptidic) cationic component of the polymericcarrier represents a cationic or polycationic polymer, typicallyexhibiting a molecular weight of about 0.1 or 0.5 kDa to about 100 kDa,preferably of about 1 kDa to about 75 kDa, more preferably of about 5kDa to about 50 kDa, even more preferably of about 5 kDa to about 30kDa, or a molecular weight of about 10 kDa to about 50 kDa, even morepreferably of about 10 kDa to about 30 kDa. Additionally, the(non-peptidic) cationic or polycationic polymer typically exhibits atleast one —SH-moiety, which is capable to form a disulfide linkage uponcondensation with either other cationic components or other componentsof the polymeric carrier as defined herein.

In the above context, the (non-peptidic) cationic component of thepolymeric carrier may be selected from acrylates, modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), chitosanes,aziridines or 2-ethyl-2-oxazoline (forming oligo ethylenimines ormodified oligoethylenimines), polymers obtained by reaction ofbisacrylates with amines forming oligo beta aminoesters or poly amidoamines, or other polymers like polyesters, polycarbonates, etc. Eachmolecule of these (non-peptidic) cationic or polycationic polymerstypically exhibits at least one —SH-moiety, wherein these at least one—SH-moiety may be introduced into the (non-peptidic) cationic orpolycationic polymer by chemical modifications, e.g. using imonothiolan,3-thio propionic acid or introduction of —SH-moieties containing aminoacids, such as cysteine or any further (modified) amino acid. Such—SH-moieties are preferably as already defined above.

In the context of the polymeric carrier, the cationic components, whichform basis for the polymeric carrier by disulfide-crosslinkage, may bethe same or different from each other. It is also particularly preferredthat the polymeric carrier of the present invention comprises mixturesof cationic peptides, proteins or polymers and optionally furthercomponents as defined herein, which are crosslinked by disulfide bondsas described herein.

In this context, the inventive polymeric carrier cargo complex due toits variable polymeric carrier advantageously allows to combine desiredproperties of different (short) cationic or polycationic peptides,proteins or polymers or other components. The polymeric carrier, e.g.,allows to efficiently compact nucleic acids for the purpose of efficienttransfection of nucleic acids, for adjuvant therapy, for the purposes ofgene therapy, for gene knock-down or others strategies without loss ofactivity, particularly exhibiting an efficient transfection of a nucleicacid into different cell lines in vitro but particularly transfection invivo. The polymeric carrier and thus the inventive polymeric carriercargo complex is furthermore not toxic to cells, provides for efficientrelease of its nucleic acid cargo, is stable during lyophilization andis applicable as immunostimulating agent or adjuvant. Preferably, thepolymer carrier cargo complex may induce the anti-viral cytokineIFN-alpha.

In particular, the polymeric carrier formed by disulfide-linked cationiccomponents allows considerably to vary its peptide or polymeric contentand thus to modulate its biophysical/biochemical properties,particularly the cationic properties of the polymeric carrier, quiteeasily and fast, e.g. by incorporating as cationic components the sameor different cationic peptide(s) or polymer(s) and optionally addingother components into the polymeric carrier. Even though consisting ofquite small non-toxic monomer units the polymeric carrier forms a longcationic binding sequence providing a strong condensation of the nucleicacid cargo and complex stability. Under the reducing conditions of thecytosol (e.g. cytosolic GSH), the complex is rapidly degraded into its(cationic) components, which are further degraded (e.g. intooligopeptides). This supports the liberation of the nucleic acid cargoin the cytosol. Due to degradation into small oligopeptides or polymersin the cytosol, no toxicity is observed as known for high-molecularoligopeptides or polymers, e.g. from high-molecular polyarginine.

Accordingly, the polymeric carrier of the inventive polymeric carriercargo complex may comprise different (short) cationic or polycationicpeptides, proteins or polymers selected from cationic or polycationicpeptides, proteins or (non-peptidic) polymers as defined above,optionally together with further components as defined herein.

Additionally, the polymeric carrier of the inventive polymeric carriercargo complex as defined above, more preferably at least one of thedifferent (short) cationic or polycationic peptides or (non-peptidic)polymers forming basis for the polymeric carrier viadisulfide-crosslinking, may be, preferably prior to thedisulfide-crosslinking, be modified with at least one further component.Alternatively, the polymeric carrier as such may be modified with atleast one further component. It may also optionally comprise at leastone further component, which typically forms the polymeric carrierdisulfide together with the other the (short) cationic or polycationicpeptides as defined above via disulfide crosslinking.

To allow modification of a cationic or polycationic peptide or a(non-peptidic) polymer as defined above, each of the components of thepolymeric carrier may (preferably already prior todisulfide-crosslinking) also contain at least one further functionalmoiety, which allows attaching such further components as definedherein. Such functional moieties may be selected from functionalitieswhich allow the attachment of further components, e.g. functionalitiesas defined herein, e.g. by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc.), by Michael addition (e.g maleinimidemoieties, α,β unsatured carbonyls, etc.), by click chemistry (e.g.azides or alkines), by alkene/alkine methatesis (e.g. alkenes oralkines), imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components.

According to a particularly preferred embodiment, the further component,which may be contained in the polymeric carrier or which may be used tomodify the different (short) cationic or polycationic peptides or(non-peptidic) polymers forming basis for the polymeric carrier of theinventive polymeric carrier cargo complex is an amino acid component(AA), which may e.g. modify the biophysical/biochemical properties ofthe polymeric carrier as defined herein. According to the presentinvention, the amino acid component (AA) comprises a number of aminoacids preferably in a range of about 1 to 100, preferably in a range ofabout 1 to 50, more preferably selected from a number comprising 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-20, or may be selectedfrom a range formed by any two of the afore mentioned values. In thiscontext the amino acids of amino acid component (AA) can be chosenindependently from each other. For example if in the polymeric carriertwo or more (AA) components are present they can be the same or can bedifferent from each other.

The amino acid component (AA) may contain or may be flanked (e.g.terminally) by a —SH containing moiety, which allows introducing thiscomponent (AA) via a disulfide bond into the polymeric carrier asdefined herein. In the specific case that the —SH containing moietyrepresents a cysteine, the amino acid component (AA) may also be read as-Cys-(AA)-Cys- wherein Cys represents Cysteine and provides for thenecessary —SH-moiety for a disulfide bond. The —SH containing moiety maybe also introduced into amino acid component (AA) using any ofmodifications or reactions as shown above for the cationic component orany of its components.

Furthermore, the amino acid component (AA) may be provided with two—SH-moieties (or even more), e.g. in a form represented by formulaHS-(AA)-SH to allow binding to two functionalities via disulfide bonds,e.g. if the amino acid component (AA) is used as a linker between twofurther components (e.g. as a linker between two cationic polymers). Inthis case, one —SH moiety is preferably protected in a first step usinga protecting group as known in the art, leading to an amino acidcomponent (AA) of formula HS-(AA)-S-protecting group. Then, the aminoacid component (AA) may be bound to a further component of the polymericcarrier, to form a first disulfide bond via the non-protected —SHmoiety. The protected-SH-moiety is then typically deprotected and boundto a further free —SH-moiety of a further component of the polymericcarrier to form a second disulfide bond.

Alternatively, the amino acid component (AA) may be provided with otherfunctionalities as already described above for the other components ofthe polymeric carrier, which allow binding of the amino acid component(AA) to any of components of the polymeric carrier.

Thus, according to the present invention, the amino acid component (AA)may be bound to further components of the polymeric carrier with orwithout using a disulfide linkage. Binding without using a disulfidelinkage may be accomplished by any of the reactions described above,preferably by binding the amino acid component (AA) to the othercomponent of the polymeric carrier using an amid-chemistry as definedherein. If desired or necessary, the other terminus of the amino acidcomponent (AA), e.g. the N- or C-terminus, may be used to couple anothercomponent, e.g. a ligand L. For this purpose, the other terminus of theamino acid component (AA) preferably comprises or is modified tocomprise a further functionality, e.g. an alkyn-species (see above),which may be used to add the other component via e.g. click-chemistry.If the ligand is bound via an acid-labile bond, the bond is preferablycleaved off in the endosome and the polymeric carrier presents aminoacid component (AA) at its surface.

The amino acid component (AA) may occur as a further component of thepolymeric carrier as defined above, e.g. as a linker between cationiccomponents e.g. as a linker between one cationic peptide and a furthercationic peptide, as a linker between one cationic polymer and a furthercationic polymer, as a linker between one cationic peptide and acationic polymer, all preferably as defined herein, or as an additionalcomponent of the polymeric carrier, e.g. by binding the amino acidcomponent (AA) to the polymeric carrier or a component thereof, e.g. viaside chains, SH-moieties or via further moieties as defined herein,wherein the amino acid component (AA) is preferably accordinglymodified.

According to a further and particularly preferred alternative, the aminoacid component (AA), may be used to modify the polymeric carrier,particularly the content of cationic components in the polymeric carrieras defined above.

In this context it is preferable, that the content of cationiccomponents in the polymeric carrier is at least 10%, 20%, or 30%,preferably at least 40%, more preferably at least 50%, 60% or 70%, butalso preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%, or may be in the range of about 30% to 100%,more preferably in the range of about 50% to 100%, even preferably inthe range of about 70% to 100%, e.g. 70, 80, 90 or 100%, or in a rangeformed by any two of the afore mentioned values, provided, that thecontent of all components in the polymeric carrier is 100%.

In the context of the present invention, the amino acid component (AA)may be selected from the following alternatives.

According to a first alternative, the amino acid component (AA) may bean aromatic amino acid component (AA). The incorporation of aromaticamino acids or sequences as amino aromatic acid component (AA) into thepolymeric carrier of the present invention enables a different (second)binding of the polymeric carrier to the nucleic acid due to interactionsof the aromatic amino acids with the bases of the nucleic acid cargo incontrast to the binding thereof by cationic charged sequences of thepolymeric carrier molecule to the phosphate backbone. This interactionmay occur e.g. by intercalations or by minor or major groove binding.This kind of interaction is not prone to decompaction by anioniccomplexing partners (e.g. Heparin, Hyaluronic acids) which are foundmainly in the extracellular matrix in vivo and is also less susceptibleto salt effects.

For this purpose, the amino acids in the aromatic amino acid component(AA) may be selected from either the same or different aromatic aminoacids e.g. selected from Trp, Tyr, or Phe. Alternatively, the aminoacids (or the entire aromatic amino acid component (AA)) may be selectedfrom following peptide combinations Trp-Tyr, Tyr-Trp, Trp-Trp, Tyr-Tyr,Trp-Tyr-Trp, Tyr-Trp-Tyr, Trp-Trp-Trp, Tyr-Tyr-Tyr, Trp-Tyr-Trp-Tyr,Tyr-Trp-Tyr-Trp, Trp-Trp-Trp-Trp, Phe-Tyr, Tyr-Phe, Phe-Phe,Phe-Tyr-Phe, Tyr-Phe-Tyr, Phe-Phe-Phe, Phe-Tyr-Phe-Tyr, Tyr-Phe-Tyr-Phe,Phe-Phe-Phe-Phe, Phe-Trp, Trp-Phe, Phe-Phe, Phe-Trp-Phe, Trp-Phe-Trp,Phe-Trp-Phe-Trp, Trp-Phe-Trp-Phe, or Tyr-Tyr-Tyr-Tyr, etc. (SEQ ID NOs:15-42). Such peptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 13, 14, 15 or even more times. These peptidecombinations may also be combined with each other as suitable.

Additionally, the aromatic amino acid component (AA) may contain or maybe flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of the polymericcarrier as defined above, e.g. as a linker. Such a —SH containing moietymay be any moiety as defined herein suitable to couple one component asdefined herein to a further component as defined herein. As an example,such a —SH containing moiety may be a cysteine. Then, e.g. the aromaticamino acid component (AA) may be selected from e.g. peptide combinationsCys-Tyr-Cys, Cys-Trp-Cys, Cys-Trp-Tyr-Cys, Cys-Tyr-Trp-Cys,Cys-Trp-Trp-Cys, Cys-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Cys,Cys-Tyr-Trp-Tyr-Cys, Cys-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Cys,Cys-Trp-Tyr-Trp-Tyr-Cys, Cys-Tyr-Trp-Tyr-Trp-Cys,Cys-Trp-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Tyr-Cys, Cys-Phe-Cys,Cys-Phe-Tyr-Cys, Cys-Tyr-Phe-Cys, Cys-Phe-Phe-Cys, Cys-Tyr-Tyr-Cys,Cys-Phe-Tyr-Phe-Cys, Cys-Tyr-Phe-Tyr-Cys, Cys-Phe-Phe-Phe-Cys,Cys-Tyr-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Tyr-Cys, Cys-Tyr-Phe-Tyr-Phe-Cys,or Cys-Phe-Phe-Phe-Phe-Cys, Cys-Phe-Trp-Cys, Cys-Trp-Phe-Cys,Cys-Phe-Phe-Cys, Cys-Phe-Trp-Phe-Cys, Cys-Trp-Phe-Trp-Cys,Cys-Phe-Trp-Phe-Trp-Cys, Cys-Trp-Phe-Trp-Phe-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein. (SEQ ID NOs: 43-75) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the aromatic amino acid component (AA) may contain orrepresent at least one proline, which may serve as a structure breakerof longer sequences of Trp, Tyr and Phe in the aromatic amino acidcomponent (AA), preferably two, three or more prolines.

According to a second alternative, the amino acid component (AA) may bea hydrophilic (and preferably non charged polar) amino acid component(AA). The incorporation of hydrophilic (and preferably non chargedpolar) amino acids or sequences as amino hydrophilic (and preferably noncharged polar) acid component (AA) into the polymeric carrier of thepresent invention enables a more flexible binding to the nucleic acidcargo. This leads to a more effective compaction of the nucleic acidcargo and hence to a better protection against nucleases and unwanteddecompaction. It also allows provision of a (long) polymeric carrierwhich exhibits a reduced cationic charge over the entire carrier and inthis context to better adjusted binding properties, if desired ornecessary.

For this purpose, the amino acids in the hydrophilic (and preferably noncharged polar) amino acid component (AA) may be selected from either thesame or different hydrophilic (and preferably non charged polar) aminoacids e.g. selected from Thr, Ser, Asn or Gln. Alternatively, the aminoacids (or the entire hydrophilic (and preferably non charged polar)amino acid component (AA)) may be selected from following peptidecombinations Ser-Thr, Thr-Ser, Ser-Ser, Thr-Thr, Ser-Thr-Ser,Thr-Ser-Thr, Ser-Ser-Ser, Thr-Thr-Thr, Ser-Thr-Ser-Thr, Thr-Ser-Thr-Ser,Ser-Ser-Ser-Ser, Thr-Thr-Thr-Thr, Gln-Asn, Asn-Gln, Gln-Gln, Asn-Asn,Gln-Asn-Gln, Asn-Gln-Asn, Gln-Gln-Gln, Asn-Asn-Asn, Gln-Asn-Gln-Asn,Asn-Gln-Asn-Gln, Gln-Gln-Gln-Gln, Asn-Asn-Asn-Asn, Ser-Asn, Asn-Ser,Ser-Ser, Asn-Asn, Ser-Asn-Ser, Asn-Ser-Asn, Ser-Ser-Ser, Asn-Asn-Asn,Ser-Asn-Ser-Asn, Asn-Ser-Asn-Ser, Ser-Ser-Ser-Ser, or Asn-Asn-Asn-Asn,etc. (SEQ ID NOs: 76-111). Such peptide combinations may be repeatede.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or even more times.These peptide combinations may also be combined with each other assuitable.

Additionally, the hydrophilic (and preferably non-charged polar) aminoacid component (AA) may contain or may be flanked by a —SH containingmoiety, which allows introducing this component via a disulfide bond asa further part of generic formula (I) above, e.g. as a linker. Such a—SH containing moiety may be any moiety as defined herein suitable tocouple one component as defined herein to a further component as definedherein. As an example, such a —SH containing moiety may be a cysteine.Then, e.g. the hydrophilic (and preferably non-charged polar) amino acidcomponent (AA) may be selected from e.g. peptide combinationsCys-Thr-Cys, Cys-Ser-Cys, Cys-Ser-Thr-Cys, Cys-Thr-Ser-Cys,Cys-Ser-Ser-Cys, Cys-Thr-Thr-Cys, Cys-Ser-Thr-Ser-Cys,Cys-Thr-Ser-Thr-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Cys,Cys-Ser-Thr-Ser-Thr-Cys, Cys-Thr-Ser-Thr-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Thr-Cys, Cys-Asn-Cys,Cys-Gln-Cys, Cys-Gln-Asn-Cys, Cys-Asn-Gln-Cys, Cys-Gln-Gln-Cys,Cys-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Cys, Cys-Asn-Gln-Asn-Cys,Cys-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Asn-Cys,Cys-Asn-Gln-Asn-Gln-Cys, Cys-Gln-Gln-Gln-Gln-Cys,Cys-Asn-Asn-Asn-Asn-Cys, Cys-Asn-Cys, Cys-Ser-Cys, Cys-Ser-Asn-Cys,Cys-Asn-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Cys,Cys-Asn-Ser-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Cys,Cys-Ser-Asn-Ser-Asn-Cys, Cys-Asn-Ser-Asn-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, or Cys-Asn-Asn-Asn-Asn-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein (SEQ ID NOs: 112-153). Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the hydrophilic (and preferably non-charged polar) aminoacid component (AA) may contain at least one proline, which may serve asa structure breaker of longer sequences of Ser, Thr and Asn in thehydrophilic (and preferably non charged polar) amino acid component(AA), preferably two, three or more prolines.

According to a third alternative, the amino acid component (AA) may be alipohilic amino acid component (AA). The incorporation of lipohilicamino acids or sequences as amino lipohilic acid component (AA) into thepolymeric carrier of the present invention enables a stronger compactionof the nucleic acid cargo and/or the polymeric carrier and its nucleicacid cargo when forming a complex. This is particularly due tointeractions of one or more polymer strands of the polymeric carrier,particularly of lipophilic sections of lipohilic amino acid component(AA) and the nucleic acid cargo. This interaction will preferably add anadditional stability to the complex between the polymeric carrier andits nucleic acid cargo. This stabilization may somehow be compared to asort of non covalent crosslinking between different polymer strands.Especially in aqueous environment this interaction is typically strongand provides a significant effect.

For this purpose, the amino acids in the lipophilic amino acid component(AA) may be selected from either the same or different lipophilic aminoacids e.g. selected from Leu, Val, Ile, Ala, Met. Alternatively, theamino acid AA (or the entire lipophilic amino acid component (AA)) maybe selected from following peptide combinations Leu-Val, Val-Leu,Leu-Leu, Val-Val, Leu-Val-Leu, Val-Leu-Val, Leu-Leu-Leu, Val-Val-Val,Leu-Val-Leu-Val, Val-Leu-Val-Leu, Leu-Leu-Leu-Leu, Val-Val-Val-Val,Ile-Ala, Ala-Ile, Ile-Ile, Ala-Ala, Ile-Ala-Ile, Ala-Ile-Ala,Ile-Ile-Ile, Ala-Ala-Ala, Ile-Ala-Be-Ala, Ala-Ile-Ala-Ile,Ile-Ile-Ile-Ile, Ala-Ala-Ala-Ala, Met-Ala, Ala-Met, Met-Met, Ala-Ala,Met-Ala-Met, Ala-Met-Ala, Met-Met-Met, Ala-Ala-Ala, Met-Ala-Met-Ala,Ala-Met-Ala-Met, or Met-Met-Met-Met etc. (SEQ ID NOs: 154-188). Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the lipophilic amino acid component (AA) may contain ormay be flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of the polymericcarrier above, e.g. as a linker. Such a —SH containing moiety may be anymoiety as defined herein suitable to couple one component as definedherein to a further component as defined herein. As an example, such a—SH containing moiety may be a cysteine. Then, e.g. the lipophilic aminoacid component (AA) may be selected from e.g. peptide combinationsCys-Val-Cys, Cys-Leu-Cys, Cys-Leu-Val-Cys, Cys-Val-Leu-Cys,Cys-Leu-Leu-Cys, Cys-Val-Val-Cys, Cys-Leu-Val-Leu-Cys,Cys-Val-Leu-Val-Cys, Cys-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Cys,Cys-Leu-Val-Leu-Val-Cys, Cys-Val-Leu-Val-Leu-Cys,Cys-Leu-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Val-Cys, Cys-Ala-Cys,Cys-Ile-Cys, Cys-Ile-Ala-Cys, Cys-Ala-Ile-Cys, Cys-Ile-Ile-Cys,Cys-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Cys, Cys-Ala-Ile-Ala-Cys,Cys-Ile-Ile-Ile-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Ala-Cys,Cys-Ala-Ile-Ala-Ile-Cys, Cys-Ile-Ile-Ile-Ile-Cys, orCys-Ala-Ala-Ala-Ala-Cys, Cys-Met-Cys, Cys-Met-Ala-Cys, Cys-Ala-Met-Cys,Cys-Met-Met-Cys, Cys-Ala-Ala-Cys, Cys-Met-Ala-Met-Cys,Cys-Ala-Met-Ala-Cys, Cys-Met-Met-Met-Cys, Cys-Ala-Ala-Ala-Cys,Cys-Met-Ala-Met-Ala-Cys, Cys-Ala-Met-Ala-Met-Cys,Cys-Met-Met-Met-Met-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein. (SEQ ID NOs: 189-229).Such peptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 13, 14, 15 or even more times. These peptide combinations mayalso be combined with each other as suitable.

Additionally, the lipophilic amino acid component (AA) may contain atleast one proline, which may serve as a structure breaker of longersequences of Leu, Val, Ile, Ala and Met in the lipophilic amino acidcomponent (AA), preferably two, three or more prolines.

Finally, according to a fourth alternative, the amino acid component(AA) may be a weak basic amino acid component (AA). The incorporation ofweak basic amino acids or sequences as weak basic amino acid component(AA) into the polymeric carrier of the present invention may serve as aproton sponge and facilitates endosomal escape (also called endosomalrelease) (proton sponge effect). Incorporation of such a weak basicamino acid component (AA) preferably enhances transfection efficiency.

For this purpose, the amino acids in the weak basic amino acid component(AA) may be selected from either the same or different weak amino acidse.g. selected from histidine or aspartate (aspartic acid).Alternatively, the weak basic amino acids (or the entire weak basicamino acid component (AA)) may be selected from following peptidecombinations Asp-His, His-Asp, Asp-Asp, His-His, Asp-His-Asp,His-Asp-His, Asp-Asp-Asp, His-His-His, Asp-His-Asp-His, His-Asp-His-Asp,Asp-Asp-Asp-Asp, or His-His-His-His, etc. (SEQ ID NOs: 230-241). Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the weak basic amino acid component (AA) may contain ormay be flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of generic formula (I)above, e.g. as a linker. Such a —SH containing moiety may be any moietyas defined herein suitable to couple one component as defined herein toa further component as defined herein. As an example, such a —SHcontaining moiety may be a cysteine. Then, e.g. the weak basic aminoacid component (AA) may be selected from e.g. peptide combinationsCys-His-Cys, Cys-Asp-Cys, Cys-Asp-His-Cys, Cys-His-Asp-Cys,Cys-Asp-Asp-Cys, Cys-His-His-Cys, Cys-Asp-His-Asp-Cys,Cys-His-Asp-His-Cys, Cys-Asp-Asp-Asp-Cys, Cys-His-His-His-Cys,Cys-Asp-His-Asp-His-Cys, Cys-His-Asp-His-Asp-Cys,Cys-Asp-Asp-Asp-Asp-Cys, or Cys-His-His-His-His-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein (SEQ ID NOs: 242-255). Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the weak basic amino acid component (AA) may contain atleast one proline, which may serve as a structure breaker of longersequences of histidine or aspartate (aspartic acid) in the weak basicamino acid component (AA), preferably two, three or more prolines.

According to a fifth alternative, the amino acid component (AA) may be asignal peptide or signal sequence, a localization signal or sequence, anuclear localization signal or sequence (NLS), an antibody, a cellpenetrating peptide, (e.g. TAT), etc. Preferably such an amino acidcomponent (AA) is bound to the polymeric carrier or to another componentof the polymeric carrier via a (reversible) disulfide bond. In thiscontext the signal peptide or signal sequence, a localization signal orsequence, a nuclear localization signal or sequence (NLS), an antibody,a cell penetrating peptide, (e.g. TAT), etc.; additionally comprises atleast one —SH-moiety. In this context a signal peptide, a localizationsignal or sequence or a nuclear localization signal or sequence (NLS),may be used to direct the inventive polymeric carrier cargo complex tospecific target cells (e.g. hepatocytes or antigen-presenting cells) andpreferably allows a translocalization of the polymeric carrier to aspecific target, e.g. into the cell, into the nucleus, into theendosomal compartment, sequences for the mitochondrial matrix,localisation sequences for the plasma membrane, localisation sequencesfor the Golgi apparatus, the nucleus, the cytoplasm and thecytosceleton, etc. Such signal peptide, a localization signal orsequence or a nuclear localization signal may be used for the transportof any of the herein defined nucleic acids, preferably an RNA or a DNA,more preferably an shRNA or a pDNA, e.g. into the nucleus. Without beinglimited thereto, such a signal peptide, a localization signal orsequence or a nuclear localization signal may comprise, e.g.,localisation sequences for the endoplasmic reticulum. Particularlocalization signals or sequences or a nuclear localization signals mayinclude e.g. KDEL (SEQ ID NO: 256), DDEL (SEQ ID NO: 257), DEEL (SEQ IDNO: 258), QEDL (SEQ ID NO: 259), RDEL (SEQ ID NO: 260), and GQNLSTSN(SEQ ID NO: 261), nuclear localisation sequences, including PKKKRKV (SEQID NO: 262), PQKKIKS (SEQ ID NO: 263), QPKKP (SEQ ID NO: 264), RKKR (SEQID NO: 265), RKKRRQRRRAHQ (SEQ ID NO: 266), RQARRNRRRRWRERQR (SEQ ID NO:267), MPLTRRRPAASQALAPPTP (SEQ ID NO: 268), GAALTILV (SEQ ID NO: 269),and GAALTLLG (SEQ ID NO: 270), localisation sequences for the endosomalcompartment, including MDDQRDLISNNEQLP (SEQ ID NO: 271), localisationsequences for the mitochondrial matrix, includingMLFNLRXXLNNAAFRHGHNFMVRNFRCGQPLX (SEQ ID NO: 272), localisationsequences for the plasma membrane: GCVCSSNP (SEQ ID NO: 273), GQTVTTPL(SEQ ID NO: 274), GQELSQHE (SEQ ID NO: 275), GNSPSYNP (SEQ ID NO: 276),GVSGSKGQ (SEQ ID NO: 277), GQTITTPL (SEQ ID NO: 278), GQTLTTPL (SEQ IDNO: 279), GQIFSRSA (SEQ ID NO: 280), GQIHGLSP (SEQ ID NO: 281), GARASVLS(SEQ ID NO: 282), and GCTLSAEE (SEQ ID NO: 283), localisation sequencesfor the endoplasmic reticulum and the nucleus, including GAQVSSQK (SEQID NO: 284), and GAQLSRNT (SEQ ID NO: 285), localisation sequences forthe Golgi apparatus, the nucleus, the cytoplasm and the cytosceleton,including GNAAAAKK (SEQ ID NO: 286), localisation sequences for thecytoplasm and cytosceleton, including GNEASYPL (SEQ ID NO: 287),localisation sequences for the plasma membrane and cytosceleton,including GSSKSKPK (SEQ ID NO: 288), etc. Examples of secretory signalpeptide sequences as defined herein include, without being limitedthereto, signal sequences of classical or non-classical MHC-molecules(e.g. signal sequences of MHC I and II molecules, e.g. of the MHC classI molecule HLA-A*0201), signal sequences of cytokines or immunoglobulinsas defined herein, signal sequences of the invariant chain ofimmunoglobulins or antibodies as defined herein, signal sequences ofLamp1, Tapasin, Erp57, Calreticulin, Calnexin, and further membraneassociated proteins or of proteins associated with the endoplasmicreticulum (ER) or the endosomal-lysosomal compartment. Particularlypreferably, signal sequences of MHC class I molecule HLA-A*0201 may beused according to the present invention. Such an additional componentmay be bound e.g. to a cationic polymer or to any other component of thepolymeric carrier as defined herein. Preferably this signal peptide,localization signal or sequence or nuclear localization signal orsequence (NLS), is bound to the polymeric carrier or to anothercomponent of the polymeric carrier via a (reversible) disulfide bond.For this purpose the (AA) component additionally comprises at least one—SH moiety as defined herein. The binding to any of components of thepolymeric carrier may also be accomplished using an acid-labile bond,preferably via a side chain of any of components of the polymericcarrier, which allows to detach or release the additional component atlower pH-values, e.g. at physiological pH-values as defined herein.

Additionally, according to another alternative, the amino acid component(AA) may be a functional peptide or protein, which may modulate thefunctionality of the polymeric carrier accordingly. Such functionalpeptides or proteins as the amino acid component (AA) preferablycomprise any peptides or proteins as defined herein, e.g. as definedbelow as therapeutically active proteins. According to one alternative,such further functional peptides or proteins may comprise so called cellpenetrating peptides (CPPs) or cationic peptides for transportation.Particularly preferred are CPPs, which induce a pH-mediatedconformational change in the endosome and lead to an improved release ofthe polymeric carrier (in complex with a nucleic acid) from the endosomeby insertion into the lipid layer of the liposome. These cellpenetrating peptides (CPPs) or cationic peptides for transportation, mayinclude, without being limited thereto protamine, nucleoline, spermineor spermidine, oligo- or poly-L-lysine (PLL), basic polypeptides, oligoor poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, suchas Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat(HIV), Tat-derived peptides, members of the penetratin family, e.g.Penetratin, Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, pIs1, etc., antimicrobial-derived CPPs e.g.Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP,MAP, KALA, PpTG20, Loligomere, FGF, Lactoferrin, histones, VP22 derivedor analog peptides, HSV, VP22 (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs, PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitoninpeptide(s), etc. Such an amino acid component (AA) may also be bound toany component of the polymeric carrier as defined herein. Preferably itis bound to the polymeric carrier or to another component of thepolymeric carrier via a (reversible) disulfide bond. For the abovepurpose, the amino acid component (AA) preferably comprises at least one—SH moiety as defined herein. The binding to any of components of thepolymeric carrier may also be accomplished using an SH-moiety or anacid-labile bond, preferably via a side chain of any of components ofthe polymeric carrier which allows to detach or release the additionalcomponent at lower pH-values, e.g. at physiological pH-values as definedherein.

According to a last alternative, the amino acid component (AA) mayconsist of any peptide or protein which can execute any favorablefunction in the cell. Particularly preferred are peptides or proteinsselected from therapeutically active proteins or peptides, fromantigens, e.g. tumour antigens, pathogenic antigens (animal antigens,viral antigens, protozoal antigens, bacterial antigens, allergicantigens), autoimmune antigens, or further antigens, from allergens,from antibodies, from immunostimulatory proteins or peptides, fromantigen-specific T-cell receptors, or from any other protein or peptidesuitable for a specific (therapeutic) application as defined below forcoding nucleic acids. Particularly preferred are peptide epitopes fromantigens as defined herein.

The polymeric carrier may comprise at least one of the above mentionedcationic or polycationic peptides, proteins or polymers or furthercomponents, e.g. (AA), wherein any of the above alternatives may becombined with each other, and may be formed by polymerizing same in acondensation polymerization reaction via their —SH-moieties.

According to another aspect, the polymeric carrier of the inventivepolymeric carrier cargo complex or single components thereof, e.g. ofthe above mentioned cationic or polycationic peptides, proteins orpolymers or further components, e.g. (AA), may be further modified witha ligand, preferably a carbohydrate, more preferably a sugar, even morepreferably mannose. Preferably this ligand is bound to the polymericcarrier or to a component of the polymeric carrier via a (reversible)disulfide bond or via Michael addition. In the case that the ligand isbound by a disulfide bond the ligand additionally comprises at least one—SH-moiety. These ligands may be used to direct the inventive polymericcarrier cargo complex to specific target cells (e.g. hepatocytes orantigen-presenting cells). In this context mannose is particularpreferred as ligand in the case that dendritic cells are the targetespecially for vaccination or adjuvant purposes.

According to a further embodiment of the invention, the inventivepolymeric carrier cargo complex may comprise (AA) components as definedabove which do not comprise —SH moieties. These (AA) components can beadded before or during the complexation reaction of the at least onenucleic acid molecule. Thereby, the (AA) component(s) is/are(non-covalently) incorporated into the inventive polymeric carrier cargocomplex without inclusion of the (AA) component(s) in the polymericcarrier itself by (covalent) polymerization.

According to one specific embodiment, the entire inventive polymericcarrier cargo complex may be formed by a polymerization or condensation(of at least one) of the above mentioned cationic or polycationicpeptides, proteins or polymers or further components, e.g. (AA), viatheir —SH-moieties in a first step and complexing the first nucleic acidto such a polymeric carrier in a second step. The polymeric carrier maythus contain a number of at least one or even more of the same ordifferent of the above defined cationic or polycationic peptides,proteins or polymers or further components, e.g. (AA), the numberpreferably determined by the above range.

According to one alternative specific embodiment, the inventivepolymeric carrier cargo complex is formed by carrying out thepolymerization or condensation of at least one of the above mentionedcationic or polycationic peptides, proteins or polymers or furthercomponents, e.g. (AA), via their —SH-moieties simultaneously tocomplexing the nucleic acid cargo to the (in situ prepared) polymericcarrier. Likewise, the polymeric carrier may thus also here contain anumber of at least one or even more of the same or different of theabove defined cationic or polycationic peptides, proteins or polymers orfurther components, e.g. (AA), the number preferably determined by theabove range.

The inventive polymeric carrier cargo complex additionally comprises asa cargo at least one first nucleic acid molecule. In the context of thepresent invention, such a first nucleic acid molecule may be anysuitable nucleic acid, selected e.g. from any (single-stranded ordouble-stranded) DNA, preferably, without being limited thereto, e.g.genomic DNA, single-stranded DNA molecules, double-stranded DNAmolecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, a(short) DNA oligonucleotide ((short) oligodesoxyribonucleotides), viralDNA, or may be selected e.g. from any PNA (peptide nucleic acid) or maybe selected e.g. from any (single-stranded or double-stranded) RNA,preferably, without being limited thereto, a (short) RNA oligonucleotide((short) oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), aviral RNA, replicons, an immunostimulatory RNA, a small interfering RNA(siRNA), an antisense RNA, a micro RNA, a small nuclear RNA (snRNA), asmall-hairpin (sh) RNA or riboswitches, ribozymes or aptamers; etc. Thenucleic acid molecule of the inventive polymeric carrier cargo complexmay also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messengerRNA (mRNA), or a viral RNA (vRNA). Preferably, the nucleic acid moleculeof the inventive polymeric carrier cargo complex is an RNA. Morepreferably, the nucleic acid molecule of the inventive polymeric carriercargo complex is a (linear) single-stranded RNA, even more preferably anmRNA or an immunostimulatory RNA. In the context of the presentinvention, an mRNA is typically an RNA, which is composed of severalstructural elements, e.g. an optional 5′-CAP structure, an optional5′-UTR region, an upstream positioned ribosomal binding site followed bya coding region, an optional 3′-UTR region, which may be followed by apoly-A tail (and/or a poly-C-tail). An mRNA may occur as a mono-, di-,or even multicistronic RNA, i.e. a RNA which carries the codingsequences of one, two or more proteins or peptides. Such codingsequences in di-, or even multicistronic mRNA may be separated by atleast one IRES sequence, e.g. as defined herein.

Furthermore, the nucleic acid of the inventive polymeric carrier cargocomplex may be a single- or a double-stranded nucleic acid molecule or apartially double-stranded or partially single stranded nucleic acid,which are at least partially self complementary (both of these partiallydouble-stranded or partially single stranded nucleic acid molecules aretypically formed by a longer and a shorter single-stranded nucleic acidmolecule or by two single stranded nucleic acid molecules, which areabout equal in length, wherein one single-stranded nucleic acid moleculeis in part complementary to the other single-stranded nucleic acidmolecule and both thus form a double-stranded nucleic acid molecule inthis region, i.e. a partially double-stranded or partially singlestranded nucleic acid molecule. Preferably, the nucleic acid moleculemay be a single-stranded nucleic acid molecule. Furthermore, the nucleicacid molecule may be a circular or linear nucleic acid molecule,preferably a linear nucleic acid molecule.

According to one alternative, the first nucleic acid molecule of theinventive polymeric carrier cargo complex may be a coding nucleic acid,e.g. a DNA or RNA. Moreover, the polymeric carrier cargo complex isadministered in combination with at least one second nucleic acidmolecule, which encodes a protein or a peptide.

According to one embodiment, the at least one first nucleic acidmolecule and the at least one second nucleic acid molecule are bothcoding nucleic acid molecules. Preferably, the at least one first andthe at least one second nucleic acid molecule each encode a differentpeptide or protein. In one embodiment, the first nucleic acid moleculehas a sequence, which is distinct from the sequence of the secondnucleic acid molecule, which is administered in combination with thepolymeric carrier cargo complex. Alternatively, the first nucleic acidmolecule and the second nucleic acid molecule may comprise the samesequence or be identical.

In the case of the at least one first nucleic acid molecule and/or ofthe second nucleic acid molecule, such a coding DNA or RNA may be anyDNA or RNA as defined herein. Preferably, such a coding DNA or RNA maybe a single- or a double-stranded DNA or RNA, more preferably asingle-stranded DNA or RNA, and/or a circular or linear DNA or RNA, morepreferably a linear DNA or RNA. Furthermore such a coding DNA or RNA maybe a genomic DNA, a viral RNA or DNA, a replicon, a plasmid DNA or anmRNA. Even more preferably, the coding DNA or RNA may be a (linear)single-stranded DNA or RNA. Most preferably, the nucleic acid moleculeaccording to the present invention may be a linear single-strandedmessenger RNA (mRNA). Such an mRNA may occur as a mono-, di-, or evenmulticistronic RNA, i.e. an RNA which carries the coding sequences ofone, two or more proteins or peptides. Such coding sequences in di-, oreven multicistronic mRNA may be separated by at least one IRES sequence,e.g. as defined herein.

In a preferred embodiment, the at least one second nucleic acid moleculeencodes a therapeutically active protein or an antigen as definedherein. In a particularly preferred embodiment, the at least one secondnucleic acid molecule, which is administered in combination with thepolymeric carrier cargo complex, encodes a peptide or a protein, whichis capable of eliciting an immune response, preferably an adaptiveimmune response, after administration, especially intramuscularadministration, to a host. Alternatively, the at least one secondnucleic acid molecule encodes a therapeutically active peptide orprotein.

Coding Nucleic Acids:

The at least one first nucleic acid molecule of the inventive polymericcarrier cargo complex and/or the at least one second nucleic acidmolecule, which is administered together with the polymeric carriercargo complex, may encode a protein or a peptide, which may be selected,without being restricted thereto, e.g. from therapeutically activeproteins or peptides, including adjuvant proteins, from antigens, e.g.tumour antigens, pathogenic antigens (e.g. selected, from animalantigens, from viral antigens, from protozoal antigens, from bacterialantigens), allergenic antigens, autoimmune antigens, or furtherantigens, from allergens, from antibodies, from immunostimulatoryproteins or peptides, from antigen-specific T-cell receptors, or fromany other protein or peptide suitable for a specific (therapeutic)application, wherein the coding nucleic acid may be transported into acell, a tissue or an organism and the protein may be expressedsubsequently in this cell, tissue or organism.

a) Therapeutically Active Proteins

In the context of the present invention, therapeutically active proteinsor peptides may be encoded by the first nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex and/or by thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex. Therapeutically active proteins aredefined herein as proteins which have an effect on healing, preventprophylactically or treat therapeutically a disease, preferably asdefined herein, or are proteins of which an individual is in need of.These may be selected from any naturally or synthetically designedoccurring recombinant or isolated protein known to a skilled person fromthe prior art. Without being restricted thereto therapeutically activeproteins may comprise proteins, capable of stimulating or inhibiting thesignal transduction in the cell, e.g. cytokines, lymphokines, monokines,growth factors, receptors, signal transduction molecules, transcriptionfactors, etc; anticoagulants; antithrombins; antiallergic proteins;apoptotic factors or apoptosis related proteins, therapeutic activeenzymes and any protein connected with any acquired disease or anyhereditary disease. A therapeutically active protein, which may beencoded by the first nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex and/or by the second nucleicacid molecule administered in combination with the polymeric carriercargo complex, may also be an adjuvant protein. In this context, anadjuvant protein is preferably to be understood as any protein, which iscapable to elicit an innate immune response as defined herein.Preferably, such an innate immune response comprises activation of apattern recognition receptor, such as e.g. a receptor selected from theToll-like receptor (TLR) family, including e.g. a Toll like receptorselected from human TLR1 to TLR10 or from murine Toll like receptorsTLR1 to TLR13. More preferably, the adjuvant protein is selected fromhuman adjuvant proteins or from pathogenic adjuvant proteins, selectedfrom the group consisting of, without being limited thereto, bacterialproteins, protozoan proteins, viral proteins, or fungal proteins, animalproteins, in particular from bacterial adjuvant proteins. In addition,nucleic acids encoding human proteins involved in adjuvant effects (e.g.ligands of pattern recognition receptors, pattern recoginitionreceptors, proteins of the signal transduction pathways, transcriptionfactors or cytokines) may be used as well.

b) Antigens

The first nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex mayalternatively encode an antigen. According to the present invention, theterm “antigen” refers to a substance which is recognized by the immunesystem and is capable of triggering an antigen-specific immune response,e.g. by formation of antibodies or antigen-specific T-cells as part ofan adaptive immune response. In this context, the first step of anadaptive immune response is the activation of naïve antigen-specific Tcells by antigen-presenting cells. This occurs in the lymphoid tissuesand organs through which naïve T cells are constantly passing. The threecell types that can serve as antigen-presenting cells are dendriticcells, macrophages, and B cells. Each of these cells has a distinctfunction in eliciting immune responses. Tissue dendritic cells take upantigens by phagocytosis and macropinocytosis and are stimulated byinfection to migrate to the local lymphoid tissue, where theydifferentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents to express MHC class II molecules. The unique ability of B cellsto bind and internalize soluble protein antigens via their receptors maybe important to induce T cells. By presenting the antigen on MHCmolecules leads to activation of T cells which induces theirproliferation and differentiation into armed effector T cells. The mostimportant function of effector T cells is the killing of infected cellsby CD8+ cytotoxic T cells and the activation of macrophages by TH1 cellswhich together make up cell-mediated immunity, and the activation of Bcells by both TH2 and TH1 cells to produce different classes ofantibody, thus driving the humoral immune response. T cells recognize anantigen by their T cell receptors which does not recognize and bindantigen directly, but instead recognize short peptide fragments e.g. ofpathogens' protein antigens, which are bound to MHC molecules on thesurfaces of other cells.

T cells fall into two major classes that have different effectorfunctions. The two classes are distinguished by the expression of thecell-surface proteins CD4 and CD8. These two types of

T cells differ in the class of MHC molecule that they recognize. Thereare two classes of MHC molecules—MHC class I and MHC class IImolecules—which differ in their structure and expression pattern ontissues of the body. CD4⁺ T cells bind to a MHC class II molecule andCD8⁺ T cells to a MHC class I molecule. MHC class I and MHC class IImolecules have distinct distributions among cells that reflect thedifferent effector functions of the T cells that recognize them. MHCclass I molecules present peptides from pathogens, commonly viruses toCD8⁺ T cells, which differentiate into cytotoxic T cells that arespecialized to kill any cell that they specifically recognize. Almostall cells express MHC class I molecules, although the level ofconstitutive expression varies from one cell type to the next. But notonly pathogenic peptides from viruses are presented by MHC class Imolecules, also self-antigens like tumour antigens are presented bythem. MHC class I molecules bind peptides from proteins degraded in thecytosol and transported in the endoplasmic reticulum. Thereby MHC classI molecules on the surface of cells infected with viruses or othercytosolic pathogens display peptides from these pathogen. The CD8⁺ Tcells that recognize MHC class I:peptide complexes are specialized tokill any cells displaying foreign peptides and so rid the body of cellsinfected with viruses and other cytosolic pathogens. The main functionof CD4⁺ T cells (CD4⁺ helper T cells) that recognize MHC class IImolecules is to activate other effector cells of the immune system. ThusMHC class II molecules are normally found on B lymphocytes, dendriticcells, and macrophages, cells that participate in immune responses, butnot on other tissue cells.

Macrophages, for example, are activated to kill the intravesicularpathogens they harbour, and B cells to secrete immunoglobulins againstforeign molecules. MHC class II molecules are prevented from binding topeptides in the endoplasmic reticulum and thus MHC class II moleculesbind peptides from proteins which are degraded in endosomes. They cancapture peptides from pathogens that have entered the vesicular systemof macrophages, or from antigens internalized by immature dendriticcells or the immunoglobulin receptors of B cells. Pathogens thataccumulate in large numbers inside macrophage and dendritic cellvesicles tend to stimulate the differentiation of TH1 cells, whereasextracellular antigens tend to stimulate the production of TH2 cells.TH1 cells activate the microbicidal properties of macrophages and induceB cells to make IgG antibodies that are very effective of opsonisingextracellular pathogens for ingestion by phagocytic cells, whereas TH2cells initiate the humoral response by activating naïve B cells tosecrete IgM, and induce the production of weakly opsonising antibodiessuch as IgG1 and IgG3 (mouse) and IgG2 and IgG4 (human) as well as IgAand IgE (mouse and human).

In the context of the present invention, antigens as encoded by thefirst nucleic acid molecule of the herein defined inventive polymericcarrier cargo complex and/or by the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complextypically comprise any antigen, antigenic epitope or antigenic peptide,falling under the above definition, more preferably protein and peptideantigens, e.g. tumour antigens, allergenic antigens, auto-immuneself-antigens, pathogenic antigens, etc. In particular antigens asencoded by the nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex and/or by the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex may be antigens generated outside the cell, more typicallyantigens not derived from the host organism (e.g. a human) itself (i.e.non-self antigens) but rather derived from host cells outside the hostorganism, e.g. viral antigens, bacterial antigens, fungal antigens,protozoological antigens, animal antigens, allergenic antigens, etc.Allergenic antigens (allergy antigens) are typically antigens, whichcause an allergy in a human and may be derived from either a human orother sources. Additionally, antigens as encoded by the first nucleicacid molecule of the herein defined inventive polymeric carrier cargocomplex and/or by the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex may be furthermoreantigens generated inside the cell, the tissue or the body. Suchantigens include antigens derived from the host organism (e.g. a human)itself, e.g. tumour antigens, self-antigens or auto-antigens, such asauto-immune self-antigens, etc., but also (non-self) antigens as definedherein, which have been originally been derived from host cells outsidethe host organism, but which are fragmented or degraded inside the body,tissue or cell, e.g. by (protease) degradation, metabolism, etc.

One class of antigens as encoded by the first nucleic acid molecule ofthe herein defined inventive polymeric carrier cargo complex and/or bythe second nucleic acid molecule administered in combination with thepolymeric carrier cargo complex comprises tumour antigens. “Tumourantigens” are preferably located on the surface of the (tumour) cell.Tumour antigens may also be selected from proteins, which areoverexpressed in tumour cells compared to a normal cell. Furthermore,tumour antigens also include antigens expressed in cells which are(were) not themselves (or originally not themselves) degenerated but areassociated with the supposed tumour. Antigens which are connected withtumour-supplying vessels or (re)formation thereof, in particular thoseantigens which are associated with neovascularization, e.g. growthfactors, such as VEGF, bFGF etc., are also included herein. Antigensconnected with a tumour furthermore include antigens from cells ortissues, typically embedding the tumour. Further, some substances(usually proteins or peptides) are expressed in patients suffering(knowingly or not-knowingly) from a cancer disease and they occur inincreased concentrations in the body fluids of said patients. Thesesubstances are also referred to as “tumour antigens”, however they arenot antigens in the stringent meaning of an immune response inducingsubstance. The class of tumour antigens can be divided further intotumour-specific antigens (TSAs) and tumour-associated-antigens (TAAs).TSAs can only be presented by tumour cells and never by normal “healthy”cells. They typically result from a tumour specific mutation. TAAs,which are more common, are usually presented by both tumour and healthycells. These antigens are recognized and the antigen-presenting cell canbe destroyed by cytotoxic T cells. Additionally, tumour antigens canalso occur on the surface of the tumour in the form of, e.g., a mutatedreceptor. In this case, they can be recognized by antibodies. Particularpreferred tumour antigens are selected from the group consisting of 5T4,707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-l-integrin,alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme Aracemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m,BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20,CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m,CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein,collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B,CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam,EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1,GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V,gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL,HLA-A*0201-R17I, HLA-Ai i/m, HLA-A2/m, HNE, homeobox NKX3.1,HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT,iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor,kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1,K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6,MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4,MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2,MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1,MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen,MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m,NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m,NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP,OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minorbcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF,Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin,survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1,TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b,TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR1, VEGFR-2/FLK-1, and WT1.Such tumour antigens preferably may be selected from the groupconsisting of MAGE-A1 (e.g. MAGE-A1 according to accession numberM77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accessionnumber NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according toaccession number NM_005511), GP100 (e.g. GP100 according to accessionnumber M77348), tyrosinase (e.g. tyrosinase according to accessionnumber NM_000372), surviving (e.g. survivin according to accessionnumber AF077350), CEA (e.g. CEA according to accession numberNM_004363), Her-2/neu (e.g. Her-2/neu according to accession numberM11730), WT1 (e.g. WT1 according to accession number NM_000378), PRAME(e.g. PRAME according to accession number NM_006115), EGFRI (epidermalgrowth factor receptor 1) (e.g. EGFRI (epidermal growth factorreceptor 1) according to accession number AF288738), MUC1, mucin-1 (e.g.mucin-1 according to accession number NM_002456), SEC61G (e.g. SEC61Gaccording to accession number NM_014302), hTERT (e.g. hTERT accessionnumber NM_198253), 5T4 (e.g. 5T4 according to accession numberNM_006670), NY-Eso-1 (e.g. NY-Eso1 according to accession numberNM_001327), TRP-2 (e.g. TRP-2 according to accession number NM_001922),STEAP, PCA, PSA, PSMA, etc.

According to another alternative, one further class of antigens asencoded by the first nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex and/or by the second nucleicacid molecule administered in combination with the polymeric carriercargo complex comprises allergenic antigens. Such allergenic antigensmay be selected from antigens derived from different sources, e.g. fromanimals, plants, fungi, bacteria, etc. Sources in this context includee.g. grasses, grass pollens, tree pollens, flower pollens, herb pollens,animals, dust mite, food, molds, animal venom (e.g. insect venom),drugs, or numerous environmental triggers, etc. Allergenic antigenstypically belong to different classes of compounds, such as nucleicacids and their fragments, proteins or peptides and their fragments,carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc. Ofparticular interest in the context of the present invention areantigens, which may be encoded by the first nucleic acid molecule of theinventive polymeric carrier cargo complex and/or by the second nucleicacid molecule administered in combination with the polymeric carriercargo complex, i.e. protein or peptide antigens and their fragments orepitopes, or nucleic acids and their fragments, particularly nucleicacids and their fragments, encoding such protein or peptide antigens andtheir fragments or epitopes.

According to another alternative, one further class of antigens asencoded by the first nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex and/or by the second nucleicacid molecule administered in combination with the polymeric carriercargo complex comprises antigens from a pathogen associated with aninfectious disease. Preferably, the pathogen is a viral, bacterial,fungal or protozoan pathogen. In this context particularly preferred areantigens from the pathogens Acinetobacter baumannii, Anaplasma genus,Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostomaduodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides,Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis,Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis,Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi,Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi,Bunyaviridae family, Burkholderia cepacia and other Burkholderiaspecies, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridaefamily, Campylobacter genus, Candida albicans, Candida spp, Chlamydiatrachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJDprion, Clonorchis sinensis, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, Clostridium perfringens, Clostridiumspp, Clostridium tetani, Coccidioides spp, coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus,Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4),Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichiachaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica,Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie Avirus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus(EBV), Escherichia coli O157:H7, O111 and O104:H4, Fasciola hepatica andFasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,Francisella tularensis, Fusobacterium genus, Geotrichum candidum,Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis BVirus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Herpessimplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV(Human immunodeficiency virus), Hortaea werneckii, Human bocavirus(HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7),Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus,Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassavirus, Legionella pneumophila, Leishmania genus, Leptospira genus,Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV),Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Naegleria fowleri, Necator americanus, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp,Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family,Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani,Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystisjirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV),Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsiaprowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fevervirus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus,Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus,Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcusgenus, Staphylococcus genus, Streptococcus agalactiae, Streptococcuspneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taeniagenus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocaracanis or Toxocara cati, Toxoplasma gondii, Treponema pallidum,Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuristrichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasmaurealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV),Variola major or Variola minor, vCJD prion, Venezuelan equineencephalitis virus, Vibrio cholerae, West Nile virus, Western equineencephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersiniaenterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

In specific embodiments according to the present invention, followingantigens of pathogens associated with infectious disease areparticularly preferred:

-   -   The Hemagglutinin (HA), the Neuraminidase (NA), the        Nucleoprotein (NP), the M1 protein, the M2 protein, the NS1        protein, the NS2 protein (the NEP protein: nuclear export        protein), the PA protein, the PB1 protein (polymerase basic 1        protein), the PB1-F2 protein and the PB2 protein in each case of        of Influenza virus;    -   the nucleoprotein (N), the phosphoprotein (P), the matrix        protein (M), the glycoprotein (G), and the viral RNA polymerase        (L), in each case of Rabies virus;    -   the Hepatitis B surface antigen (HBsAg), the Hepatitis B core        antigen (HbcAg), the Hepatitis B virus DNA polymerase, the HBx        protein, the preS2 middle surface protein, the large S protein,        the virus protein VP1, the virus protein VP2, the virus protein        VP3, and the virus protein VP4, in each case of Hepatitis B        virus;    -   the E1 protein, the E2 protein, the E3 protein, the E4 protein,        the E5 protein, the E6 protein, the E7 protein, the E8 protein,        the L1 protein, and the L2 protein, in each case of human        Papilloma virus (hPV);    -   the protective antigen (PA), the edema factor (EF), the lethal        factor (LF), and the S-layer homology proteins (SLH), in each        case of Bacillus anthracis;    -   the Fusion (F) protein, the nucleocapsid (N) protein, the        phosphoprotein (P), the matrix (M) protein, the glycoprotein        (G), the large protein (L; RNA polymerase), the non-structural        protein 1 (NS1), the non-structural protein 2 (NS2), the small        hydrophobic (SH) protein, the elongation factor M2-1, and the        transcription regulation protein M2-2, in each case of        respiratory syncytial virus (RSV);    -   the Glycoprotein L (UL1), the Uracil-DNA glycosylase UL2, the        UL3 protein, the UL4 protein, the DNA replication protein UL5,        the Portal protein UL6, the Virion maturation protein UL7, the        DNA helicase ULB, the Replication origin-binding protein UL9,        the Glycoprotein M (UL10), the UL11 protein, the Alkaline        exonuclease UL12, the Serine-threonine protein kinase UL13, the        Tegument protein UL14, the Terminase (UL15), the Tegument        protein UL16, the UL17 protein, the Capsid protein VP23 (UL18),        the Major capsid protein VP5 (UL19), the Membrane protein UL20,        the Tegument protein UL21, the Glycoprotein H (UL22), the        Thymidine Kinase UL23, the UL24 protein, the UL25 protein, the        Capsid protein P40 (UL26, VP24, VP22A), the Glycoprotein B        (UL27), the ICP18.5 protein (UL28), the Major DNA-binding        protein ICP8 (UL29), the DNA polymerase UL30, the Nuclear matrix        protein UL31, the Envelope glycoprotein UL32, the UL33 protein,        the Inner nuclear membrane protein UL34, the Capsid protein VP26        (UL35), the Large tegument protein UL36, the Capsid assembly        protein UL37, the VP19C protein (UL38), the Ribonucleotide        reductase (Large subunit) UL39, the Ribonucleotide reductase        (Small subunit) UL40, the Tegument protein/Virion host shutoff        VHS protein (UL41), the DNA polymerase processivity factor UL42,        the Membrane protein UL43, the Glycoprotein C (UL44), the        Membrane protein UL45, the Tegument proteins VP11/12 (UL46), the        Tegument protein VP13/14 (UL47), the Virion maturation protein        VP16 (UL48, Alpha-TIF), the Envelope protein UL49, the dUTP        diphosphatase UL50, the Tegument protein UL51, the DNA        helicase/primase complex protein UL52, the Glycoprotein K        (UL53), the Transcriptional regulation protein 1E63 (ICP27,        UL54), the UL55 protein, the UL56 protein, the Viral replication        protein ICP22 (1E68, US1), the US2 protein, the        Serine/threonine-protein kinase US3, the Glycoprotein G (US4),        the Glycoprotein J (US5), the Glycoprotein D (US6), the        Glycoprotein I (US7), the Glycoprotein E (US8), the Tegument        protein US9, the Capsid/Tegument protein US10, the Vmw21 protein        (US11), the ICP47 protein (IE12, US12), the Major        transcriptional activator ICP4 (IE175, RS1), the E3 ubiquitin        ligase ICP0 (IE110), the Latency-related protein 1 (LRP1), the        Latency-related protein 2 (LRP2), the Neurovirulence factor RL1        (ICP34.5), and the Latency-associated transcript (LAT), in each        case of Herpes simplex virus (HSV);    -   the ESAT-6 protein, the ESX-1 protein, the CFP10 protein, the        TB10.4 protein, the MPT63 protein, the MPT64 protein, the MPT83        protein, the MTB12 protein, the MTB8 protein, the AG85A protein,        the AG85B protein, the Rpf-like proteins, the KATG protein, the        PPE18 protein, the MTB32 protein, the MTB39 protein, the        Crystallin, the HSP65 protein, the PST-S protein, and the HBHA        protein, the 10 kDa filtrate antigen EsxB, the serine protease        PepA, the fibronectin-binding protein D FbpD, the secreted        protein MPT51, the periplasmic phosphate-binding lipoprotein        PSTS1 (PBP-1), the periplasmic phosphate-binding lipoprotein        PSTS3 (PBP-3, Phos-1), the PPE family protein PPE14, the PPE        family protein PPE68, the protein MTB72F, the molecular        chaperone DnaK, the cell surface lipoprotein MPT83, the        lipoprotein P23, the Phosphate transport system permease protein        PstA, the 14 kDa antigen, the fibronectin-binding protein C        FbpC1, the Alanine dehydrogenase TB43, and the Glutamine        synthetase 1, in each case of Mycobacterium tuberculosis;    -   the capsid protein C, the membrane protein M, the envelope        protein E; the nonstructural protein NS1, the nonstructural        protein NS2a, the nonstructural protein, the nonstructural        protein NS2b, the nonstructural protein NS3, the nonstructural        protein NS4a, the nonstructural protein NS4b, and the        nonstructural protein NS5 in each case of Dengue virus;    -   the structural protein VP1, the structural protein VP2, the        structural protein VP3, the structural protein VP4, the        structural protein VP6, the structural protein VP7, the        nonstructural protein NSP1, the nonstructural protein NSP2, the        nonstructural protein NSP3, the nonstructural protein NSP4, the        nonstructural protein NSP5 and the nonstructural protein NSP6 in        each case of Rotavirus;    -   the HIV p24 antigen, the HIV envelope proteins (Gp120, Gp41,        Gp160), the polyprotein GAG, the negative factor protein Nef,        the trans-activator of transcription Tat in each case of HIV        (Human immunodeficiency virus); or    -   the glycoprotein GP, the nucleoprotein NP, the minor matrix        protein VP24, the major matrix protein VP40, the transcription        activator VP30, the polymerase cofactor VP35, the RNA polymerase        L in each case of Ebolavirus (EBOV) or Marburg Virus.

Of particular interest in the context of the present invention areantigens, which may be encoded by the first nucleic acid molecule of theinventive polymeric carrier cargo complex and/or by the second nucleicacid molecule administered in combination with the polymeric carriercargo complex or comprised in a pharmaceutical composition or vaccine,i.e. protein or peptide antigens and their fragments or epitopes, ornucleic acids and their fragments, particularly nucleic acids and theirfragments, encoding such protein or peptide antigens and their fragmentsor epitopes.

c) Antibodies

According to a further alternative, the first nucleic acid molecule ofthe herein defined inventive polymeric carrier cargo complex and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex may encode an antibody or an antibodyfragment. According to the present invention, such an antibody may beselected from any antibody, e.g. any recombinantly produced or naturallyoccurring antibodies, known in the art, in particular antibodiessuitable for therapeutic, diagnostic or scientific purposes, orantibodies which have been identified in relation to specific cancerdiseases. Herein, the term “antibody” is used in its broadest sense andspecifically covers monoclonal and polyclonal antibodies (includingagonist, antagonist, and blocking or neutralizing antibodies) andantibody species with polyepitopic specificity. According to theinvention, the term “antibody” typically comprises any antibody known inthe art (e.g. IgM, IgD, IgG, IgA and IgE antibodies), such as naturallyoccurring antibodies, antibodies generated by immunization in a hostorganism, antibodies which were isolated and identified from naturallyoccurring antibodies or antibodies generated by immunization in a hostorganism and recombinantly produced by biomolecular methods known in theart, as well as chimeric antibodies, human antibodies, humanizedantibodies, bispecific antibodies, intrabodies, i.e. antibodiesexpressed in cells and optionally localized in specific cellcompartments, and fragments and variants of the aforementionedantibodies. In general, an antibody consists of a light chain and aheavy chain both having variable and constant domains. The light chainconsists of an N-terminal variable domain, V_(L), and a C-terminalconstant domain, C_(L). In contrast, the heavy chain of the IgGantibody, for example, is comprised of an N-terminal variable domain,V_(H), and three constant domains, C_(H)1, C_(H)2 and C_(H)3.

In the context of the present invention, antibodies as encoded by thefirst nucleic acid molecule of the herein defined inventive polymericcarrier cargo complex and/or by the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex maypreferably comprise full-length antibodies, i.e. antibodies composed ofthe full heavy and full light chains, as described above. However,derivatives of antibodies such as antibody fragments, variants oradducts may also be encoded by the first nucleic acid molecule of theherein defined inventive inventive polymeric carrier cargo complexand/or by the second nucleic acid molecule administered in combinationwith the polymeric carrier cargo complex. Antibody fragments arepreferably selected from Fab, Fab′, F(ab′)₂, Fc, Facb, pFc′, Fd and Fvfragments of the aforementioned (full-length) antibodies. In general,antibody fragments are known in the art. For example, a Fab (“fragment,antigen binding”) fragment is composed of one constant and one variabledomain of each of the heavy and the light chain. The two variabledomains bind the epitope on specific antigens. The two chains areconnected via a disulfide linkage. A scFv (“single chain variablefragment”) fragment, for example, typically consists of the variabledomains of the light and heavy chains. The domains are linked by anartificial linkage, in general a polypeptide linkage such as a peptidecomposed of 15-25 glycine, proline and/or serine residues.

In the present context it is preferable that the different chains of theantibody or antibody fragment are encoded by a multicistronic nucleicacid molecule. Alternatively, the different strains of the antibody orantibody fragment are encoded by several monocistronic nucleic acid(s)(sequences).

siRNA:

According to a further alternative, the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may be in theform of dsRNA, preferably siRNA. A dsRNA, or a siRNA, is of interestparticularly in connection with the phenomenon of RNA interference. Thein vitro technique of RNA interference (RNAi) is based ondouble-stranded RNA molecules (dsRNA), which trigger thesequence-specific suppression of gene expression (Zamore (2001) Nat.Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490: Hannon(2002) Nature 41: 244-251). In the transfection of mammalian cells withlong dsRNA, the activation of protein kinase R and RnaseL brings aboutunspecific effects, such as, for example, an interferon response (Starket al. (1998) Annu. Rev. Biochem. 67: 227-264; He and Katze (2002) ViralImmunol. 15: 95-119). These unspecific effects are avoided when shorter,for example 21- to 23-mer, so-called siRNA (small interfering RNA), isused, because unspecific effects are not triggered by siRNA that isshorter than 30 bp (Elbashir et al. (2001) Nature 411: 494-498).

The nucleic acid molecule of the herein defined inventive polymericcarrier cargo complex may thus be a double-stranded RNA (dsRNA) having alength of from 17 to 29, preferably from 19 to 25, and preferably is atleast 90%, more preferably 95% and especially 100% (of the nucleotidesof a dsRNA) complementary to a section of the nucleic acid molecule of a(therapeutically relevant) protein or antigen described (as activeingredient) hereinbefore or of any further protein as described herein,either a coding or a non-coding section, preferably a coding section.Such a (section of the) nucleic acid molecule may be termed herein a“target sequence” and may be any nucleic acid molecule as definedherein, preferably a genomic DNA, a cDNA, a RNA, e.g. an mRNA, etc. 90%complementary means that with a length of a dsRNA described herein of,for example, 20 nucleotides, the dsRNA contains not more than 2nucleotides showing no complementarity with the corresponding section ofthe target sequence. The sequence of the double-stranded RNA usedaccording to the invention is, however, preferably wholly complementaryin its general structure with a section of the target sequence. In thiscontext the nucleic acid molecule of the inventive polymeric carriercargo complex may be a dsRNA having the general structure5′-(N₁₇₋₂₉)-3′, preferably having the general structure 5′-(N₁₉₋₂₅)-3′,more preferably having the general structure 5′-(N₁₉₋₂₄)-3′, or yet morepreferably having the general structure 5′-(N₂₁₋₂₃)-3′, wherein for eachgeneral structure each N is a (preferably different) nucleotide of asection of the target sequence, preferably being selected from acontinuous number of 17 to 29 nucleotides of a section of the targetsequence, and being present in the general structure 5′-(N₁₇₋₂₉)-3′ intheir natural order. In principle, all the sections having a length offrom 17 to 29, preferably from 19 to 25, base pairs that occur in thetarget sequence can serve for preparation of a dsRNA as defined herein.Equally, dsRNAs used as nucleic acid molecule of the inventive polymericcarrier cargo complex can also be directed against nucleotide sequencesof a (therapeutically relevant) protein or antigen described (as activeingredient) hereinbefore that do not lie in the coding region, inparticular in the 5′ non-coding region of the target sequence, forexample, therefore, against non-coding regions of the target sequencehaving a regulatory function. The target sequence of the dsRNA used asnucleic acid molecule of the inventive polymeric carrier cargo complexcan therefore lie in the translated and untranslated region of thetarget sequence and/or in the region of the control elements of aprotein or antigen described hereinbefore. The target sequence for adsRNA used as the nucleic acid molecule of the inventive polymericcarrier cargo complex can also lie in the overlapping region ofuntranslated and translated sequence; in particular, the target sequencecan comprise at least one nucleotide upstream of the start triplet ofthe coding region, e.g. of a genomic DNA, a cDNA, a RNA, or an mRNA,etc.

Immunostimulatory Nucleic Acids: a) Immunostimulatory CpG Nucleic Acids:

According to another alternative, the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may be in theform of a(n) (immunostimulatory) CpG nucleic acid, in particular CpG-RNAor CpG-DNA, which preferably induces an innate immune response. ACpG-RNA or CpG-DNA used according to the invention can be asingle-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA),a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid used according to the invention ispreferably in the form of CpG-RNA, more preferably in the form ofsingle-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG nucleicacids have a length as described above. Preferably the CpG motifs areunmethylated.

b) Immunostimulatory RNA (isRNA):

Likewise, according to a further alternative, the (immunostimulatory)nucleic acid molecule of the inventive polymeric carrier cargo complexmay be in the form of an immunostimulatory RNA (isRNA), which preferablyelicits an innate immune response. Such an immunostimulatory RNA may beany (double-stranded or single-stranded) RNA, e.g. a coding RNA, asdefined herein. In a preferred embodiment, the immunostimulatory RNA isa non-coding RNA. Preferably, the immunostimulatory RNA may be asingle-stranded, a double-stranded or a partially double-stranded RNA,more preferably a single-stranded RNA, and/or a circular or linear RNA,more preferably a linear RNA. More preferably, the immunostimulatory RNAmay be a (linear) single-stranded RNA. Even more preferably, theimmunostimulatory RNA may be a (long) (linear) single-stranded)non-coding RNA. In this context it is particular preferred that theisRNA carries a triphosphate at its 5′-end which is the case for invitro transcribed RNA. An immunostimulatory RNA may also occur as ashort RNA oligonucleotide as defined herein.

An immunostimulatory RNA as used herein may furthermore be selected fromany class of RNA molecules, found in nature or being preparedsynthetically, and which can induce an innate immune response and maysupport an adaptive immune response induced by an antigen. In thiscontext, an immune response may occur in various ways. A substantialfactor for a suitable (adaptive) immune response is the stimulation ofdifferent T-cell sub-populations. T-lymphocytes are typically dividedinto two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2(Th2) cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of the effectorproteins (cytokines) produced by them. Thus, Th1 cells assist thecellular immune response by activation of macrophages and cytotoxicT-cells. Th2 cells, on the other hand, promote the humoral immuneresponse by stimulation of B-cells for conversion into plasma cells andby formation of antibodies (e.g. against antigens). The Th1/Th2 ratio istherefore of great importance in the induction and maintenance of anadaptive immune response. In connection with the present invention, theTh1/Th2 ratio of the (adaptive) immune response is preferably shifted inthe direction towards the cellular response (Th1 response) and acellular immune response is thereby induced. According to one example,the innate immune system which may support an adaptive immune response,may be activated by ligands of Toll-like receptors (TLRs). TLRs are afamily of highly conserved pattern recognition receptor (PRR)polypeptides that recognize pathogen-associated molecular patterns(PAMPs) and play a critical role in innate immunity in mammals.Currently at least thirteen family members, designated TLR1-TLR13(Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore,a number of specific TLR ligands have been identified. It was e.g. foundthat unmethylated bacterial DNA and synthetic analogs thereof (CpG DNA)are ligands for TLR9 (Hemmi H et al. (2000) Nature 408:740-5; Bauer S etal. (2001) Proc Natl Acad Sci USA 98, 9237-42). Furthermore, it has beenreported that ligands for certain TLRs include certain nucleic acidmolecules and that certain types of RNA are immunostimulatory in asequence-independent or sequence-dependent manner, wherein these variousimmunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, orintracellular receptors such as RIG-I, MDA-5, etc. E.g. Lipford et al.determined certain G,U-containing oligoribonucleotides asimmunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280). Theimmunostimulatory G,U-containing oligoribonucleotides described byLipford et al. were believed to be derivable from RNA sources includingribosomal RNA, transfer RNA, messenger RNA, and viral RNA.

The immunostimulatory RNA (isRNA) used as the nucleic acid molecule ofthe herein defined inventive polymeric carrier cargo complex may thuscomprise any RNA sequence, which enhances an immune response in a host.Preferably, the isRNA used as the first nucleic acid molecule of thepolymeric carrier cargo complex enhances the immune response, which ispreferably an adaptive immune response elicited by a peptide or proteinencoded by the second nucleic acid molecule, preferably an mRNA, that isadministered to the host in combination with the polymeric carrier cargocomplex. The isRNA used as the first nucleic acid molecule of thepolymeric carrier cargo complex may thus comprise any RNA sequence knownto be immunostimulatory, including, without being limited thereto, RNAsequences representing and/or encoding ligands of TLRs, preferablyselected from human family members TLR1-TLR10 or murine family membersTLR1-TLR13, more preferably selected from (human) family membersTLR1-TLR10, even more preferably from TLR7 and TLR8, ligands forintracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g.Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases:two parallel ways to trigger antiviral responses. Mol. Cell 22,561-569), or any other immunostimulatory RNA sequence. Furthermore,(classes of) immunostimulatory RNA molecules, used as the nucleic acidmolecule of the inventive polymeric carrier cargo complex may includeany other RNA capable of eliciting an innate immune response. Withoutbeing limited thereto, such an immunostimulatory RNA may includeribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), andviral RNA (vRNA). Such an immunostimulatory RNA may comprise a length of1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides.

According to a particularly preferred embodiment, such immunostimulatorynucleic acid sequences is preferably RNA preferably consisting of orcomprising a nucleic acid of formula (II) or (III):

G_(l)X_(m)G_(n),  (formula (II))

wherein:

-   G is guanosine, uracil or an analogue of guanosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the above-mentioned nucleotides;-   l is an integer from 1 to 40,    -   wherein    -   when l=1 G is guanosine or an analogue thereof,    -   when l>1 at least 50% of the nucleotides are guanosine or an        analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3 X is uracil or an analogue thereof,    -   when m>3 at least 3 successive uracils or analogues of uracil        occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1 G is guanosine or an analogue thereof,    -   when n>1 at least 50% of the nucleotides are guanosine or an        analogue thereof.

C_(l)X_(m)C_(n),  (formula (III))

wherein:

-   C is cytosine, uracil or an analogue of cytosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the above-mentioned nucleotides;-   l is an integer from 1 to 40,    -   wherein    -   when l=1 C is cytosine or an analogue thereof,    -   when l>1 at least 50% of the nucleotides are cytosine or an        analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3 X is uracil or an analogue thereof,    -   when m>3 at least 3 successive uracils or analogues of uracil        occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1 C is cytosine or an analogue thereof,    -   when n>1 at least 50% of the nucleotides are cytosine or an        analogue thereof.

The nucleic acids of formula (II) or (III), which may be used thenucleic acid cargo of the inventive polymeric carrier cargo complex maybe relatively short nucleic acid molecules with a typical length ofapproximately from 5 to 100 (but may also be longer than 100 nucleotidesfor specific embodiments, e.g. up to 200 nucleotides), from 5 to 90 orfrom 5 to 80 nucleotides, preferably a length of approximately from 5 to70, more preferably a length of approximately from 8 to 60 and, morepreferably a length of approximately from 15 to 60 nucleotides, morepreferably from 20 to 60, most preferably from 30 to 60 nucleotides. Ifthe nucleic acid of the inventive nucleic acid cargo complex has amaximum length of e.g. 100 nucleotides, m will typically be <=98. Thenumber of nucleotides G in the nucleic acid of formula (II) isdetermined by 1 or n. 1 and n, independently of one another, are each aninteger from 1 to 40, wherein when 1 or n=1 G is guanosine or ananalogue thereof, and when 1 or n>1 at least 50% of the nucleotides areguanosine or an analogue thereof. For example, without implying anylimitation, when 1 or n=4 G_(l) or G_(n) can be, for example, a GUGU,GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG or GGGG, etc.; when l orn=5 G_(l) or G_(n) can be, for example, a GGGUU, GGUGU, GUGGU, UGGGU,UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG, GGUGG, GUGGG, UGGGG, or GGGGG,etc.; etc. A nucleotide adjacent to X_(m) in the nucleic acid of formula(II) according to the invention is preferably not a uracil. Similarly,the number of nucleotides C in the nucleic acid of formula (III)according to the invention is determined by l or n. l and n,independently of one another, are each an integer from 1 to 40, whereinwhen l or n=1 C is cytosine or an analogue thereof, and when l or n>1 atleast 50% of the nucleotides are cytosine or an analogue thereof. Forexample, without implying any limitation, when l or n=4, C_(l) or C_(n)can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC, CUCC,UCCC or CCCC, etc.; when l or n=5 C_(l) or C_(n) can be, for example, aCCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC, CUCUC, CCCCU, CCCUC,CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. A nucleotide adjacent to X_(m)in the nucleic acid of formula (III) according to the invention ispreferably not a uracil. Preferably, for formula (II), when l or n>1, atleast 60%, 70%, 80%, 90% or even 100% of the nucleotides are guanosineor an analogue thereof, as defined above. The remaining nucleotides to100% (when guanosine constitutes less than 100% of the nucleotides) inthe flanking sequences G_(l) and/or G_(n) are uracil or an analoguethereof, as defined hereinbefore. Also preferably, l and n,independently of one another, are each an integer from 2 to 30, morepreferably an integer from 2 to 20 and yet more preferably an integerfrom 2 to 15. The lower limit of l or n can be varied if necessary andis at least 1, preferably at least 2, more preferably at least 3, 4, 5,6, 7, 8, 9 or 10. This definition applies correspondingly to formula(III).

According to a particularly preferred embodiment, a nucleic acidaccording to any of formulas (II) or (III) above, which may be used asnucleic acid of the inventive polymeric carrier cargo complex, may beselected from a sequence consisting or comprising any of the followingsequences:

(SEQ ID NO: 289) GGUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 290)GGGGGUUUUUUUUUUGGGGG; (SEQ ID NO: 291)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 292)GUGUGUGUGUGUUUUUUUUUUUUUUUUGUGUGUGUGUGU; (SEQ ID NO: 293)GGUUGGUUGGUUUUUUUUUUUUUUUUUGGUUGGUUGGUU; (SEQ ID NO: 294)GGGGGGGGGUUUGGGGGGGG; (SEQ ID NO: 295) GGGGGGGGUUUUGGGGGGGG;(SEQ ID NO: 296) GGGGGGGUUUUUUGGGGGGG; (SEQ ID NO: 297)GGGGGGGUUUUUUUGGGGGG; (SEQ ID NO: 298) GGGGGGUUUUUUUUGGGGGG;(SEQ ID NO: 299) GGGGGGUUUUUUUUUGGGGG; (SEQ ID NO: 300)GGGGGGUUUUUUUUUUGGGG; (SEQ ID NO: 301) GGGGGUUUUUUUUUUUGGGG;(SEQ ID NO: 302) GGGGGUUUUUUUUUUUUGGG; (SEQ ID NO: 303)GGGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 304) GGGGUUUUUUUUUUUUUUGG;(SEQ ID NO: 305) GGUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 306)GUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 307) GGGGGGGGGGUUUGGGGGGGGG;(SEQ ID NO: 308) GGGGGGGGGUUUUGGGGGGGGG; (SEQ ID NO: 309)GGGGGGGGUUUUUUGGGGGGGG; (SEQ ID NO: 310) GGGGGGGGUUUUUUUGGGGGGG;(SEQ ID NO: 311) GGGGGGGUUUUUUUUGGGGGGG; (SEQ ID NO: 312)GGGGGGGUUUUUUUUUGGGGGG; (SEQ ID NO: 313) GGGGGGGUUUUUUUUUUGGGGG;(SEQ ID NO: 314) GGGGGGUUUUUUUUUUUGGGGG; (SEQ ID NO: 315)GGGGGGUUUUUUUUUUUUGGGG; (SEQ ID NO: 316) GGGGGUUUUUUUUUUUUUGGGG;(SEQ ID NO: 317) GGGGGUUUUUUUUUUUUUUGGG; (SEQ ID NO: 318)GGGUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 319) GGUUUUUUUUUUUUUUUUUUGG;(SEQ ID NO: 320) GGGGGGGGGGGUUUGGGGGGGGGG; (SEQ ID NO: 321)GGGGGGGGGGUUUUGGGGGGGGGG; (SEQ ID NO: 322) GGGGGGGGGUUUUUUGGGGGGGGG;(SEQ ID NO: 323) GGGGGGGGGUUUUUUUGGGGGGGG; (SEQ ID NO: 324)GGGGGGGGUUUUUUUUGGGGGGGG; (SEQ ID NO: 325) GGGGGGGGUUUUUUUUUGGGGGGG;(SEQ ID NO: 326) GGGGGGGGUUUUUUUUUUGGGGGG; (SEQ ID NO: 327)GGGGGGGUUUUUUUUUUUGGGGGG; (SEQ ID NO: 328) GGGGGGGUUUUUUUUUUUUGGGGG;(SEQ ID NO: 329) GGGGGGUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 330)GGGGGGUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 331) GGGGUUUUUUUUUUUUUUUUGGGG;(SEQ ID NO: 332) GGGUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 333)GUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 334)GGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 335)GGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 336)GGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 337)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 338)GGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 339)GGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGG; (SEQ ID NO: 340)GGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGG; (SEQ ID NO: 341)GGGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGGG; (SEQ ID NO: 342)GGUUUGG; (SEQ ID NO: 343) GGUUUUGG; (SEQ ID NO: 344) GGUUUUUGG;(SEQ ID NO: 345) GGUUUUUUGG; (SEQ ID NO: 346) GGUUUUUUUGG;(SEQ ID NO: 347) GGUUUUUUUUGG; (SEQ ID NO: 348) GGUUUUUUUUUGG;(SEQ ID NO: 349) GGUUUUUUUUUUGG; (SEQ ID NO: 350) GGUUUUUUUUUUUGG;(SEQ ID NO: 351) GGUUUUUUUUUUUUGG; (SEQ ID NO: 352) GGUUUUUUUUUUUUUGG;(SEQ ID NO: 353) GGUUUUUUUUUUUUUUGG; (SEQ ID NO: 354)GGUUUUUUUUUUUUUUUGG; (SEQ ID NO: 355) GGGUUUGGG; (SEQ ID NO: 356)GGGUUUUGGG; (SEQ ID NO: 357) GGGUUUUUGGG; (SEQ ID NO: 358) GGGUUUUUUGGG;(SEQ ID NO: 359) GGGUUUUUUUGGG; (SEQ ID NO: 360) GGGUUUUUUUUGGG;(SEQ ID NO: 361) GGGUUUUUUUUUGGG; (SEQ ID NO: 362) GGGUUUUUUUUUUGGG;(SEQ ID NO: 363) GGGUUUUUUUUUUUGGG; (SEQ ID NO: 364) GGGUUUUUUUUUUUUGGG;(SEQ ID NO: 365) GGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 366)GGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUU UUUUGGG;(SEQ ID NO: 367) GGGUUUUUUUUUUUUUUUGGGGGGUUUUUUUUUUUUUUUGGG;(SEQ ID NO: 368) GGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGG G;(short GU-rich, SEQ ID NO: 369) GGUUUUUUUUUUUUUUUGGG or (SEQ ID NO: 370)CCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUU UUUUCCC(SEQ ID NO: 371) CCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCC C(SEQ ID NO: 372) CCCUUUUUUUUUUUUUUUCCCCCCUUUUUUUUUUUUUUUCCCor from a sequence having at least 60%, 70%, 80%, 90%, or even 95%sequence identity with any of these sequences

According to a further particularly preferred embodiment, suchimmunostimulatory nucleic acid sequences particularly isRNA consist ofor comprise a nucleic acid of formula (IV) or (V):

(N_(u)G_(l)X_(m)G_(n)N_(v))_(a),  (formula (IV))

wherein:

-   G is guanosine (guanine), uridine (uracil) or an analogue of    guanosine (guanine) or uridine (uracil), preferably guanosine    (guanine) or an analogue thereof;-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine), or an analogue of these    nucleotides (nucleosides), preferably uridine (uracil) or an    analogue thereof;-   N is a nucleic acid sequence having a length of about 4 to 50,    preferably of about 4 to 40, more preferably of about 4 to 30 or 4    to 20 nucleic acids, each N independently being selected from    guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of these    nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein when l=1, G is guanosine (guanine) or an analogue        thereof,        -   when l>1, at least 50% of these nucleotides (nucleosides)            are guanosine (guanine) or an analogue        -   thereof;-   m is an integer and is at least 3;    -   wherein when m=3, X is uridine (uracil) or an analogue thereof,        and    -   when m>3, at least 3 successive uridines (uracils) or analogues        of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein when n=1, G is guanosine (guanine) or an analogue        thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are guanosine (guanine) or an analogue        -   thereof;-   u,v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v≥1, or        -   when v=0, u≥1;            wherein the nucleic acid molecule of formula (IV) has a            length of at least 50 nucleotides, preferably of at least            100 nucleotides, more preferably of at least 150            nucleotides, even more preferably of at least 200            nucleotides and most preferably of at least 250 nucleotides.

(N_(u)C_(l)X_(m)C_(n)N_(v))_(a),  (formula (V))

wherein:

-   C is cytidine (cytosine), uridine (uracil) or an analogue of    cytidine (cytosine) or uridine (uracil), preferably cytidine    (cytosine) or an analogue thereof;-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of the    above-mentioned nucleotides (nucleosides), preferably uridine    (uracil) or an analogue thereof;-   N is each a nucleic acid sequence having independent from each other    a length of about 4 to 50, preferably of about 4 to 40, more    preferably of about 4 to 30 or 4 to 20 nucleic acids, each N    independently being selected from guanosine (guanine), uridine    (uracil), adenosine (adenine), thymidine (thymine), cytidine    (cytosine) or an analogue of these nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein when l=1, C is cytidine (cytosine) or an analogue        thereof,        -   when l>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue        -   thereof;-   m is an integer and is at least 3;    -   wherein when m=3, X is uridine (uracil) or an analogue thereof,        -   when m>3, at least 3 successive uridines (uracils) or            analogues of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein when n=1, C is cytidine (cytosine) or an analogue        thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue        -   thereof.-   u, v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v 1, or        -   when v=0, u≥1;            wherein the nucleic acid molecule of formula (V) according            to the invention has a length of at least 50 nucleotides,            preferably of at least 100 nucleotides, more preferably of            at least 150 nucleotides, even more preferably of at least            200 nucleotides and most preferably of at least 250            nucleotides.

For formula (V), any of the definitions given above for elements N (i.e.N_(u) and N_(v)) and X (X_(m)), particularly the core structure asdefined above, as well as for integers a, l, m, n, u and v, similarlyapply to elements of formula (V) correspondingly, wherein in formula (V)the core structure is defined by C_(l)X_(m)C_(n). The definition ofbordering elements N_(u) and N_(v) is identical to the definitions givenabove for N_(u) and N_(v).

According to a very particularly preferred embodiment, the inventivenucleic acid molecule according to formula (IV) may be selected frome.g. any of the following sequences:

(SEQ ID NO: 373) UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACG (SEQ ID NO: 374)UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUCCAAAAAAAAAAAAAAACCC ACGCAAGGAUCUUCAUGUGC(SEQ ID NO: 375) GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUC (SEQ ID NO: 376)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAG (SEQ ID NO: 377)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAGAGCUACGCAGGUUCGCAAUAAAAGCGUUGAUUAGUGUGCAUAGAACAGACCUCUUAUUCGGUGAAACGCCAGAAUGCUAAAUUCCAAUAACUCUUCCCAAAACGCGUACGGCCGAAGACGCGCGCUUAUCUUGUGUACGUUCUCGCACAUGGAAGAAUCAGCGGGCAUGGUGGUAGGGCAAUAGGGGAGCUGGGUAGCAGCGAAAAAGGGCCCCUGCGCACGUAGCUUCGCUGUUCGUCUGAAACAACCCGGCAUCCGUUGUAGCGAUCCCGUUAUCAGUGUUAUUCUUGUGCGCACUAAGAUUCAUGGUGUAGUCGACAAUAACAGCGUCUUGGCAGAUUCUGGUCACGUGCCCUAUGCCCGGGCUUGUGCCUCUCAGGUGCACAGCGAUACUUAAAGCCUUCAAGGUACUCGACGUGGGUACCGAUUCGUGACACUUCCUAAGAUUAUUCCACUGUGUUAGCCCCGCACCGCCGACCUAAACUGGUCCAAUGUAUACGCAUUCGCUGAGCGGAUCGAUAAUAAAAGCUUGAAUU (SEQ ID NO: 378)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUC (SEQ ID NO: 379)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUA(R2025/R2391 SEQ ID NO: 385)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAG (SEQ ID NO: 380)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAGAACGAACUGACCUGACGCCUGAACUUAUGAGCGUGCGUAUUUUUUUUUUUUUUUUUUUUUUUCCUCCCAACAAAUGUCGAUCAAUAGCUGGGCUGUUGGAGACGCGUCAGCAAAUGCCGUGGCUCCAUAGGACGUGUAGACUUCUAUUUUUUUUUUUUUUUUUUUUUCCCGGGACCACAAAUAAUAUUCUUGCUUGGUUGGGCGCAAGGGCCCCGUAUCAGGUCAUAAACGGGUACAUGUUGCACAGGCUCCUUUUUUUUUUUUUUUUUUUUUUUCGCUGAGUUAUUCCGGUCUCAAAAGACGGCAGACGUCAGUCGACAACACGGUCUAAAGCAGUGCUACAAUCUGCCGUGUUCGUGUUUUUUUUUUUUUUUUUUUUGUGAACCUACACGGCGUGCACUGUAGUUCGCAAUUCAUAGGGUACCGGCUCAGAGUUAUGCCUUGGUUGAAAACUGCCCAGCAUACUUUUUUUUUUUUUUUUUUUUCAUAUUCCCAUGCUAAGCAAGGGAUGCCGCGAGUCAUGUUAAGCUUGAAUU

According to another very particularly preferred embodiment, the nucleicacid molecule according to formula (V) may be selected from e.g. any ofthe following sequences:

(SEQ ID NO: 381) UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACG or (SEQ ID NO: 382)UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUGGAAAAAAAAAAAAAAAGGG ACGCAAGGAUCUUCAUGUGC

In a further preferred embodiment the first nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex may also occur in the form of a modifiednucleic acid.

In this context, the first nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex and/or the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex may be provided as a “stabilized nucleic acid”, preferably as astabilized RNA or DNA, more preferably as a RNA that is essentiallyresistant to in vivo degradation (e.g. by an exo- or endo-nuclease).

Preferably, the first nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex and/or the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex may contain backbone modifications, sugar modifications or basemodifications. A backbone modification in connection with the presentinvention is a modification in which phosphates of the backbone of thenucleotides contained in the nucleic acid molecule of the inventivepolymeric carrier cargo complex are chemically modified. A sugarmodification in connection with the present invention is a chemicalmodification of the sugar of the nucleotides of the first nucleic acidmolecule of the inventive polymeric carrier cargo complex and/or of thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex. Furthermore, a base modification inconnection with the present invention is a chemical modification of thebase moiety of the nucleotides of the nucleic acid molecule of theinventive polymeric carrier cargo complex and/or of the second nucleicacid molecule administered in combination with the polymeric carriercargo complex. Such a modification preferably increases the stability ofthe nucleic acid molecule of the inventive polymeric carrier cargocomplex and/or of the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex, more preferably anRNA molecule, and/or the expression of a protein encoded by the firstand/or the second nucleic acid molecule. Several nucleic acidmodifications are known in the art, which can be applied to a nucleicacid molecule in the context of the present invention.

Chemical Modifications:

The term “nucleic acid modification” as used herein may refer tochemical modifications comprising backbone modifications as well assugar modifications or base modifications.

In this context, the nucleic acid molecule of the inventive polymericcarrier cargo complex and/or of the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex maycontain nucleotide analogues/modifications, e.g. backbone modifications,sugar modifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in a nucleicacid molecule, preferably an RNA molecule as defined herein, arechemically modified. A sugar modification in connection with the presentinvention is a chemical modification of the sugar of the nucleotides ofa nucleic acid molecule as defined herein. Furthermore, a basemodification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the nucleic acidmolecule of the inventive polymeric carrier cargo complex and/or of thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex. In this context, nucleotide analoguesor modifications are preferably selected from nucleotide analogues,which are applicable for transcription and/or translation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated intothe nucleic acid molecule of the inventive polymeric carrier cargocomplex and/or of the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex, can be modified inthe sugar moiety. For example, the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. Examples of “oxy”-2′ hydroxyl group modifications include,but are not limited to, alkoxy or aryloxy (—OR, e.g., R═H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols(PEG), —O(CH₂CH₂O)nCH₂CH₂OR; “locked” nucleic acids (LNA) in which the2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbonof the same ribose sugar; and amino groups (—O-amino, wherein the aminogroup, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl,arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylenediamine, polyamino) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH2; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, nucleic acid molecule of the inventive polymericcarrier cargo complex and/or of the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex caninclude nucleotides containing, for instance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into the nucleicacid molecule of the inventive polymeric carrier cargo complex and/or ofthe second nucleic acid molecule administered in combination with thepolymeric carrier cargo complex. The phosphate groups of the backbonecan be modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the full replacement of an unmodified phosphate moiety witha modified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intothe nucleic acid molecule of the inventive polymeric carrier cargocomplex and/or of the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex, can further bemodified in the nucleobase moiety. Examples of nucleobases found innucleic acid molecules include, but are not limited to, adenine,guanine, cytosine and uracil. For example, the nucleosides andnucleotides described herein can be chemically modified on the majorgroove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridine-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl) adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(1-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(1-Thiophosphate)-Pseudouridine.

In further specific embodiments, the nucleic acid molecule of theinventive polymeric carrier cargo complex and/or of the second nucleicacid molecule administered in combination with the polymeric carriercargo complex may comprise nucleoside modifications selected from6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, Pseudo-iso-cytidine,5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine,5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine,5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine,inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, α-thio-adenosine,8-azido-adenosine, 7-deaza-adenosine.

Modification of the 5′-End of a Modified RNA Molecule:

According to another preferred embodiment of the invention, the nucleicacid molecule of the inventive polymeric carrier cargo complex and/or ofthe second nucleic acid molecule administered in combination with thepolymeric carrier cargo complex may be an RNA molecule, preferably amodified RNA molecule as defined herein, which is modified by theaddition of a so-called “5′ CAP” structure.

A 5′-cap is an entity, typically a modified nucleotide entity, whichgenerally “caps” the 5′-end of a mature mRNA. A 5′-cap may typically beformed by a modified nucleotide, particularly by a derivative of aguanine nucleotide. Preferably, the 5′-cap is linked to the 5′-terminusvia a 5′-5′-triphosphate linkage. A 5′-cap may be methylated, e.g.m7GpppN, wherein N is the terminal 5′ nucleotide of the nucleic acidcarrying the 5′-cap, typically the 5′-end of an RNA. m7GpppN is the5′-CAP structure which naturally occurs in mRNA transcribed bypolymerase II and is therefore not considered as modification comprisedin a modified RNA in this context. According to a preferred embodiment,the nucleic acid molecule of the inventive polymeric carrier cargocomplex and/or of the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex may thus comprise am7GpppN as 5′-CAP, and preferably comprise, in addition, at least onefurther modification as defined herein.

Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-CAP structuresare regarded as at least one modification in this context.

Particularly preferred modified 5′-CAP structures are CAP1 (methylationof the ribose of the adjacent nucleotide of m7G), CAP2 (methylation ofthe ribose of the 2nd nucleotide downstream of the m7G), CAP3(methylation of the ribose of the 3rd nucleotide downstream of the m7G),CAP4 (methylation of the ribose of the 4th nucleotide downstream of them7G), ARCA (anti-reverse CAP analogue, modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

According to a further embodiment, the first nucleic acid molecule ofthe herein defined inventive polymeric carrier cargo complex and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex can contain a lipid modification. Such alipid-modified nucleic acid typically comprises a nucleic acid asdefined herein. Such a lipid-modified first nucleic acid molecule of theinventive polymeric carrier cargo complex or a lipid-modified secondnucleic acid molecule administered in combination with the polymericcarrier cargo complex typically further comprises at least one linkercovalently linked with that nucleic acid molecule, and at least onelipid covalently linked with the respective linker. Alternatively, thelipid-modified nucleic acid molecule comprises at least one nucleic acidmolecule as defined herein and at least one (bifunctional) lipidcovalently linked (without a linker) with that nucleic acid molecule.According to a third alternative, the lipid-modified nucleic acidmolecule comprises a nucleic acid molecule as defined herein, at leastone linker covalently linked with that nucleic acid molecule, and atleast one lipid covalently linked with the respective linker, and alsoat least one (bifunctional) lipid covalently linked (without a linker)with that nucleic acid molecule.

The first nucleic acid molecule of the inventive polymeric carrier cargocomplex and/or the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex may likewise bestabilized in order to prevent degradation of the nucleic acid moleculeby various approaches, particularly, when RNA or mRNA is used as anucleic acid molecule for the inventive purpose. It is known in the artthat instability and (fast) degradation of RNA in general may representa serious problem in the application of RNA based compositions. Thisinstability of RNA is typically due to RNA-degrading enzymes, “RNAases”(ribonucleases), wherein contamination with such ribonucleases maysometimes completely degrade RNA in solution. Accordingly, the naturaldegradation of RNA in the cytoplasm of cells is very finely regulatedand RNase contaminations may be generally removed by special treatmentprior to use of said compositions, in particular with diethylpyrocarbonate (DEPC). A number of mechanisms of natural degradation areknown in this connection in the prior art, which may be utilized aswell. E.g., the terminal structure is typically of critical importanceparticularly for an mRNA. As an example, at the 5′ end of naturallyoccurring mRNAs there is usually a so-called “cap structure” (a modifiedguanosine nucleotide), and at the 3′ end is typically a sequence of upto 200 adenosine nucleotides (the so-called poly-A tail).

According to another embodiment, the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex and/or the secondnucleic acid molecule administered in combination with the polymericcarrier cargo complex may be modified, and thus stabilized, especiallyif the nucleic acid molecule is in the form of a coding nucleic acide.g. an mRNA, by modifying the G/C content of the nucleic acid molecule,particularly an mRNA, preferably of the coding region thereof.

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region of the first nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex and/or of thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex, especially if the nucleic acid moleculeis in the form of an mRNA, is modified, particularly increased, comparedto the G/C content of the coding region of its particular wild typecoding sequence, i.e. the unmodified mRNA. The encoded amino acidsequence of the nucleic acid sequence is preferably not modifiedcompared to the coded amino acid sequence of the particular wild typemRNA.

The modification of the G/C-content of the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex, especially if the nucleic acid moleculeis in the form of an mRNA or codes for an mRNA, is based on the factthat the sequence of any mRNA region to be translated is important forefficient translation of that mRNA. Thus, the composition and thesequence of various nucleotides are important. In particular, sequenceshaving an increased G (guanosine)/C (cytosine) content are more stablethan sequences having an increased A (adenosine)/U (uracil) content.According to the invention, the codons of the coding sequence or mRNAare therefore varied compared to its wild type coding sequence or mRNA,while retaining the translated amino acid sequence, such that theyinclude an increased amount of G/C nucleotides. In respect to the factthat several codons code for one and the same amino acid (so-calleddegeneration of the genetic code), the most favourable codons for thestability can be determined (so-called alternative codon usage).

Preferably, the G/C content of the coding region of the nucleic acidmolecule of the herein defined inventive polymeric carrier cargo complexand/or the second nucleic acid molecule administered in combination withthe polymeric carrier cargo complex, especially if the nucleic acid isin the form of an mRNA or codes for an mRNA, is increased by at least7%, more preferably by at least 15%, particularly preferably by at least20%, compared to the G/C content of the coded region of the wild typemRNA. According to a specific aspect at least 5%, 10%, 20%, 30%, 40%,50%, 60%, more preferably at least 70%, even more preferably at least80% and most preferably at least 90%, 95% or even 100% of thesubstitutable codons in the region coding for a protein or peptide asdefined herein or its fragment or variant thereof or the whole sequenceof the wild type mRNA sequence or coding sequence are substituted,thereby increasing the G/C content of said sequence.

In this context, it is particularly preferable to increase the G/Ccontent of the nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex,especially if the nucleic acid is in the form of an mRNA or codes for anmRNA, to the maximum (i.e. 100% of the substitutable codons), inparticular in the region coding for a protein, compared to the wild typesequence.

According to the invention, a further preferred modification of thenucleic acid molecule of the herein defined inventive polymeric carriercargo complex and/or the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex, especially if thenucleic acid is in the form of an mRNA or codes for an mRNA, is based onthe finding that the translation efficiency is also determined by adifferent frequency in the occurrence of tRNAs in cells. Thus, ifso-called “rare codons” are present in the nucleic acid molecule of theinventive polymeric carrier cargo complex and/or the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex, especially if the nucleic acid is in the form of an mRNA orcodes for an mRNA, to an increased extent, the corresponding modifiednucleic acid molecule is translated to a significantly poorer degreethan in the case where codons coding for relatively “frequent” tRNAs arepresent.

Especially if the modified nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex and/or the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex is in the form of an mRNA or codes for an mRNA, the codingregion of the modified nucleic acid is preferably modified compared tothe corresponding region of the wild type mRNA or coding sequence suchthat at least one codon of the wild type sequence which codes for a tRNAwhich is relatively rare in the cell is exchanged for a codon whichcodes for a tRNA which is relatively frequent in the cell and carriesthe same amino acid as the relatively rare tRNA. By this modification,the sequences of the nucleic acid molecule of the inventive polymericcarrier cargo complex and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex,especially if the nucleic acid is in the form of an mRNA or codes for anmRNA, is modified such that codons for which frequently occurring tRNAsare available are inserted. In other words, according to the invention,by this modification all codons of the wild type sequence which code fora tRNA which is relatively rare in the cell can in each case beexchanged for a codon which codes for a tRNA which is relativelyfrequent in the cell and which, in each case, carries the same aminoacid as the relatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe modified nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex and/or the modified second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex, especially if the nucleic acid is in the form of an mRNA orcodes for an mRNA, with the “frequent” codons without modifying theamino acid sequence of the protein encoded by the coding region of thenucleic acid molecule. This preferred aspect allows provision of aparticularly efficiently translated and stabilized (modified) nucleicacid molecule, especially if the nucleic acid is in the form of an mRNAor codes for an mRNA.

According to a further preferred embodiment of the invention, thenucleic acid molecule of the inventive polymeric carrier cargo complexas defined herein and/or the second nucleic acid molecule administeredin combination with the polymeric carrier cargo complex, especially ifthe nucleic acid is in the form of a coding nucleic acid molecule,preferably has at least one 5′ and/or 3′ stabilizing sequence. Thesestabilizing sequences in the 5′ and/or 3′ untranslated regions have theeffect of increasing the half-life of the nucleic acid in the cytosol.These stabilizing sequences can have 100% sequence identity to naturallyoccurring sequences which occur in viruses, bacteria and eukaryotes, butcan also be partly or completely synthetic. The untranslated sequences(UTR) of the (alpha-)globin gene, e.g. from Homo sapiens or Xenopuslaevis may be mentioned as an example of stabilizing sequences which canbe used in the present invention for a stabilized nucleic acid. Anotherexample of a stabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 383), which iscontained in the 3′UTR of the very stable RNA which codes for(alpha-)globin, type(I)-collagen, 15-lipoxygenase or for tyrosinehydroxylase (cf. Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94:2410 to 2414). Such stabilizing sequences can of course be usedindividually or in combination with one another and also in combinationwith other stabilizing sequences known to a person skilled in the art.

In one embodiment of the invention, the nucleic acid molecule of theinventive polymeric carrier cargo complex as defined herein and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex may be an RNA molecule, which ispreferably modified as defined herein, more preferably an mRNA molecule,wherein the mRNA molecule comprises at least one selected from the groupconsisting of a 5′-UTR, a 3′-UTR, a poly(A) sequence, a poly(C) sequenceand a histone stem-loop sequence. In a particularly preferredembodiment, the second nucleic acid molecule, which is administered incombination with the polymeric carrier cargo complex is an mRNAmolecule, preferably an mRNA molecule comprising at least onemodification as defined herein, wherein the mRNA preferably comprises atleast one selected from the group consisting of a 5′-UTR, a 3′-UTR, apoly(A) sequence, a poly(C) sequence and a histone stem-loop sequence.

In a preferred embodiment, the nucleic acid molecule of the inventivepolymeric carrier cargo complex as defined herein and/or the secondnucleic acid molecule administered in combination with the polymericcarrier cargo complex comprises a 5′-UTR and/or a 3′-UTR.

In the context of the present invention, a 3′-UTR is typically the partof an mRNA, which is located between the protein coding region (i.e. theopen reading frame) and the 3′-terminus of the mRNA. A 3′-UTR of an mRNAis not translated into an amino acid sequence. The 3′-UTR sequence isgenerally encoded by the gene, which is transcribed into the respectivemRNA during the gene expression process. In the context of the presentinvention, a 3′-UTR corresponds to the sequence of a mature mRNA whichis located 3′ to the stop codon of the protein coding region, preferablyimmediately 3′ to the stop codon of the protein coding region, and whichextends to the 5′-side of the 3′-terminus of the mRNA or of the poly(A)sequence, preferably to the nucleotide immediately 5′ to the poly(A)sequence. The term “corresponds to” means that the 3′-UTR sequence maybe an RNA sequence, such as in the mRNA sequence used for defining the3′-UTR sequence, or a DNA sequence which corresponds to such RNAsequence. In the context of the present invention, the term “a 3′-UTR ofa gene”, such as “a 3′-UTR of an albumin gene”, is the sequence, whichcorresponds to the 3′-UTR of the mature mRNA derived from this gene,i.e. the mRNA obtained by transcription of the gene and maturation ofthe pre-mature mRNA. The term “3′-UTR of a gene” encompasses the DNAsequence and the RNA sequence of the 3′-UTR. Preferably, the 3′-UTR usedaccording to the present invention is heterologous to the coding regionof the mRNA sequence. Even if 3′-UTR's derived from naturally occurringgenes are preferred, also synthetically engineered UTR's may be used inthe context of the present invention.

As used herein, the term ‘5′-UTR’ typically refers to a particularsection of messenger RNA (mRNA). It is located 5′ of the open readingframe of the mRNA. Typically, the 5′-UTR starts with the transcriptionalstart site and ends one nucleotide before the start codon of the openreading frame. The 5′-UTR may comprise elements for controlling geneexpression, also called regulatory elements. Such regulatory elementsmay be, for example, ribosomal binding sites or a 5′-TerminalOligopyrimidine Tract. The 5′-UTR may be posttranscriptionally modified,for example by addition of a 5′-CAP. In the context of the presentinvention, a 5′-UTR corresponds to the sequence of a mature mRNA, whichis located between the 5′-CAP and the start codon. Preferably, the5′-UTR corresponds to the sequence, which extends from a nucleotidelocated 3′ to the 5′-CAP, preferably from the nucleotide locatedimmediately 3′ to the 5′-CAP, to a nucleotide located 5′ to the startcodon of the protein coding region, preferably to the nucleotide locatedimmediately 5′ to the start codon of the protein coding region. Thenucleotide located immediately 3′ to the 5′-CAP of a mature mRNAtypically corresponds to the transcriptional start site. The term“corresponds to” means that the 5′-UTR sequence may be an RNA sequence,such as in the mRNA sequence used for defining the 5′-UTR sequence, or aDNA sequence, which corresponds to such RNA sequence. In the context ofthe present invention, the term “a 5′-UTR of a gene”, such as “a 5′-UTRof a TOP gene”, is the sequence, which corresponds to the 5′-UTR of themature mRNA derived from this gene, i.e. the mRNA obtained bytranscription of the gene and maturation of the pre-mature mRNA. Theterm “5′-UTR of a gene” encompasses the DNA sequence and the RNAsequence of the 5′-UTR. Preferably, the 5′-UTR used according to thepresent invention is heterologous to the coding region of the mRNAsequence. Even if 5′-UTR's derived from naturally occurring genes arepreferred, also synthetically engineered UTR's may be used in thecontext of the present invention.

In a particularly preferred embodiment, the nucleic acid molecule of theinventive polymeric carrier cargo complex as defined herein and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex comprises at least one 5′-untranslatedregion (5′-UTR). More preferably, the nucleic acid molecule of theinventive polymeric carrier cargo complex as defined herein and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex comprises a 5′-UTR, which comprises orconsists of a nucleic acid sequence which is derived from a 5′-UTR of aTOP gene, or which is derived from a fragment, homolog or variant of the5′-UTR of a TOP gene.

The 5′terminal oligopyrimidine tract (TOP) is typically a stretch ofpyrimidine nucleotides located at the 5′ terminal region of a nucleicacid molecule, such as the 5′ terminal region of certain mRNA moleculesor the 5′ terminal region of a functional entity, e.g. the transcribedregion, of certain genes. The sequence starts with a cytidine, whichusually corresponds to the transcriptional start site, and is followedby a stretch of usually about 3 to 30 pyrimidine nucleotides. Forexample, the TOP may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or evenmore nucleotides. The pyrimidine stretch and thus the 5′ TOP ends onenucleotide 5′ to the first purine nucleotide located downstream of theTOP. Messenger RNA that contains a 5′terminal oligopyrimidine tract isoften referred to as TOP mRNA. Accordingly, genes that provide suchmessenger RNAs are referred to as TOP genes. TOP sequences have, forexample, been found in genes and mRNAs encoding peptide elongationfactors and ribosomal proteins.

In the context of the present invention, a TOP motif is typically anucleic acid sequence, which corresponds to a 5′TOP as defined above.Thus, a TOP motif in the context of the present invention is preferablya stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides,preferably at least 4 pyrimidine nucleotides, preferably at least 5pyrimidine nucleotides, more preferably at least 6 nucleotides, morepreferably at least 7 nucleotides, most preferably at least 8 pyrimidinenucleotides, wherein the stretch of pyrimidine nucleotides preferablystarts at its 5′ end with a cytosine nucleotide. In TOP genes and TOPmRNAs, the TOP-motif preferably starts at its 5′end with thetranscriptional start site and ends one nucleotide 5′ to the firstpurine residue in said gene or mRNA. A TOP motif in the sense of thepresent invention is preferably located at the 5′end of a sequence,which represents a 5′-UTR or at the 5′end of a sequence, which codes fora 5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidinenucleotides is called “TOP motif” in the sense of the present inventionif this stretch is located at the 5′end of a respective sequence, suchas the inventive mRNA, the 5′-UTR of the inventive mRNA, or the nucleicacid sequence, which is derived from the 5′-UTR of a TOP gene asdescribed herein. In other words, a stretch of 3 or more pyrimidinenucleotides, which is not located at the 5′-end of a 5′-UTR but anywherewithin a 5′-UTR is preferably not referred to as “TOP motif”.

In this context, a TOP gene is typically characterised by the presenceof a 5′ terminal oligopyrimidine tract. Furthermore, most TOP genes arecharacterized by a growth-associated translational regulation. However,also TOP genes with a tissue specific translational regulation areknown. As defined above, the 5′-UTR of a TOP gene corresponds to thesequence of a 5′-UTR of a mature mRNA derived from a TOP gene, whichpreferably extends from the nucleotide located 3′ to the 5′-CAP to thenucleotide located 5′ to the start codon. A 5′-UTR of a TOP genetypically does not comprise any start codons, preferably no upstreamAUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstreamAUGs and upstream open reading frames are typically understood to beAUGs and open reading frames that occur 5′ of the start codon (AUG) ofthe open reading frame that should be translated. The 5′-UTRs of TOPgenes are generally rather short. The lengths of 5′-UTRs of TOP genesmay vary between 20 nucleotides up to 500 nucleotides, and are typicallyless than about 200 nucleotides, preferably less than about 150nucleotides, more preferably less than about 100 nucleotides. Exemplary5′-UTRs of TOP genes in the sense of the present invention are thenucleic acid sequences extending from the nucleotide at position 5 tothe nucleotide located immediately 5′ to the start codon (e.g. the ATG)in the sequences according to SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQID NO. 1421 and SEQ ID NO. 1422 of the international patent applicationWO2013/143700 or homologs or variants thereof, whose disclosure isincorporated herewith by reference. In this context a particularlypreferred fragment of a 5′-UTR of a TOP gene is a 5′-UTR of a TOP genelacking the 5′TOP motif. The term ‘5′-UTR of a TOP gene’ preferablyrefers to the 5′-UTR of a naturally occurring TOP gene.

In a specific embodiment, the 5′-UTR does not comprise a TOP-motif or a5′TOP, as defined herein.

In some embodiments, the nucleic acid sequence of the 5′-UTR, which isderived from a 5′-UTR of a TOP gene terminates at its 3′-end with anucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstreamof the start codon (e.g. A(U/T)G) of the gene or mRNA it is derivedfrom. Thus, the 5′-UTR does not comprise any part of the protein codingregion. Thus, preferably, the only protein coding part of the inventivemRNA sequence is provided by the coding region.

The nucleic acid sequence, which is derived from a 5′-UTR of a TOP gene,is preferably derived from a eukaryotic TOP gene, preferably a plant oranimal TOP gene, more preferably a chordate TOP gene, even morepreferably a vertebrate TOP gene, most preferably a mammalian TOP gene,such as a human TOP gene.

For example, the 5′-UTR preferably comprises or consists of a nucleicacid sequence, which is derived from a nucleic acid sequence selectedfrom the group consisting of SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ IDNO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700,whose disclosure is incorporated herein by reference, from the homologsof SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.1422 of the patent application WO2013/143700, from a variant thereof, orpreferably from a corresponding RNA sequence. The term “homologs of SEQID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 ofthe patent application WO2013/143700” refers to sequences of otherspecies than Homo sapiens, which are homologous to the sequencesaccording to SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 andSEQ ID NO. 1422 of the patent application WO2013/143700.

In a preferred embodiment, the 5′-UTR comprises or consists of a nucleicacid sequence, which is derived from a nucleic acid sequence extendingfrom nucleotide position 5 (i.e. the nucleotide that is located atposition 5 in the sequence) to the nucleotide position immediately 5′ tothe start codon (located at the 3′ end of the sequences), e.g. thenucleotide position immediately 5′ to the ATG sequence, of a nucleicacid sequence selected from SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ IDNO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700,from the homologs of SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO.1421 and SEQ ID NO. 1422 of the patent application WO2013/143700 from avariant thereof, or a corresponding RNA sequence. It is particularlypreferred that the 5′ UTR is derived from a nucleic acid sequenceextending from the nucleotide position immediately 3′ to the 5′TOP tothe nucleotide position immediately 5′ to the start codon (located atthe 3′ end of the sequences), e.g. the nucleotide position immediately5′ to the ATG sequence, of a nucleic acid sequence selected from SEQ IDNos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700, from the homologs of SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700, from a variant thereof, or acorresponding RNA sequence.

In a particularly preferred embodiment, the 5′-UTR comprises or consistsof a nucleic acid sequence, which is derived from a 5′-UTR of aribosomal protein gene, preferably from a 5′-UTR of a TOP gene encodinga ribosomal protein or from a variant of a 5′-UTR of a TOP gene encodinga ribosomal protein. For example, the 5′-UTR comprises or consists of anucleic acid sequence, which is derived from a 5′-UTR of a nucleic acidsequence according to any of SEQ ID NOs: 67, 170, 193, 244, 259, 554,650, 675, 700, 721, 913, 1016, 1063, 1120, 1138, and 1284-1360 of thepatent application WO2013/143700, a corresponding RNA sequence, ahomolog thereof, or a variant thereof as described herein, preferablylacking the 5′TOP motif. As described above, the sequence extending fromposition 5 to the nucleotide immediately 5′ to the ATG (which is locatedat the 3′end of the sequences) corresponds to the 5′-UTR of saidsequences.

Preferably, the 5′-UTR comprises or consists of a nucleic acid sequence,which is derived from a 5′-UTR of a TOP gene encoding a ribosomal Largeprotein (RPL) or from a homolog or variant of a 5′-UTR of a TOP geneencoding a ribosomal Large protein (RPL). For example, the 5′-UTRcomprises or consists of a nucleic acid sequence, which is derived froma 5′-UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67,259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and 1422 of thepatent application WO2013/143700, a corresponding RNA sequence, ahomolog thereof, or a variant thereof as described herein, preferablylacking the 5′TOP motif.

In a particularly preferred embodiment, the 5′-UTR comprises or consistsof a nucleic acid sequence, which is derived from the 5′-UTR of aribosomal protein Large 32 gene, preferably from a vertebrate ribosomalprotein Large 32 (L32) gene, more preferably from a mammalian ribosomalprotein Large 32 (L32) gene, most preferably from a human ribosomalprotein Large 32 (L32) gene, or from a variant of the 5′-UTR of aribosomal protein Large 32 gene, preferably from a vertebrate ribosomalprotein Large 32 (L32) gene, more preferably from a mammalian ribosomalprotein Large 32 (L32) gene, most preferably from a human ribosomalprotein Large 32 (L32) gene, wherein preferably the 5′-UTR does notcomprise the 5′TOP of said gene.

A preferred sequence for a 5′-UTR element corresponds to SEQ ID NO. 1368of the patent application WO2013/143700 and reads as follows:

Nucleotide sequence for 5′-UTR element (SEQ ID NO. 386)GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC

Accordingly, in a particularly preferred embodiment, the 5′-UTRcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID NO. 1368 of thepatent application WO2013/143700 (5′-UTR of human ribosomal proteinLarge 32 lacking the 5′ terminal oligopyrimidine tract, SEQ ID NO. 32)or preferably to a corresponding RNA sequence, or wherein the at leastone 5′-UTR comprises or consists of a fragment of a nucleic acidsequence, which has an identity of at least about 40%, preferably of atleast about 50%, preferably of at least about 60%, preferably of atleast about 70%, more preferably of at least about 80%, more preferablyof at least about 90%, even more preferably of at least about 95%, evenmore preferably of at least about 99% to the nucleic acid sequenceaccording to SEQ ID NO. 31 or more preferably to a corresponding RNAsequence, wherein, preferably, the fragment is as described above, i.e.being a continuous stretch of nucleotides representing at least 20% etc.of the full-length 5′-UTR. Preferably, the fragment exhibits a length ofat least about 20 nucleotides or more, preferably of at least about 30nucleotides or more, more preferably of at least about 40 nucleotides ormore. Preferably, the fragment is a functional fragment as describedherein.

In some embodiments, the inventive mRNA sequence comprises a 5′-UTR,which comprises or consists of a nucleic acid sequence, which is derivedfrom the 5′-UTR of a vertebrate TOP gene, such as a mammalian, e.g. ahuman TOP gene, selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6,RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A,RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26,RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A,RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15,RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26,RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A,RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40, RPL41, RPLP0, RPLP1,RPLP2, RPLP3, RPLP0, RPLP1, RPLP2, EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2,EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1,HNRNPA1, TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2, UQCRB, or froma homolog or variant thereof, wherein preferably the 5′-UTR does notcomprise a TOP-motif or the 5′TOP of said genes, and wherein optionallythe 5′-UTR starts at its 5′-end with a nucleotide located at position 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 downstream of the 5′terminaloligopyrimidine tract (TOP) and wherein further optionally the 5′-UTR,which is derived from a 5′-UTR of a TOP gene, terminates at its 3′-endwith a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10upstream of the start codon (A(U/T)G) of the gene it is derived from.

In further particularly preferred embodiments, the 5′-UTR comprises orconsists of a nucleic acid sequence, which is derived from the 5′-UTR ofa ribosomal protein Large 32 gene (RPL32), a ribosomal protein Large 35gene (RPL35), a ribosomal protein Large 21 gene (RPL21), an ATPsynthase, H+transporting, mitochondrial Fl complex, alpha subunit 1,cardiac muscle (ATP5A1) gene, an hydroxysteroid (17-beta) dehydrogenase4 gene (HSD17B4), an androgen-induced 1 gene (AIG1), cytochrome coxidase subunit VIc gene (COX6C), or a N-acylsphingosine amidohydrolase(acid ceramidase) 1 gene (ASAH1) or from a variant thereof, preferablyfrom a vertebrate ribosomal protein Large 32 gene (RPL32), a vertebrateribosomal protein Large 35 gene (RPL35), a vertebrate ribosomal proteinLarge 21 gene (RPL21), a vertebrate ATP synthase, H+ transporting,mitochondrial F1 complex, alpha subunit 1, cardiac muscle (ATP5A1) gene,a vertebrate hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), avertebrate androgen-induced 1 gene (AIG1), a vertebrate cytochrome coxidase subunit VIc gene (COX6C), or a vertebrate N-acylsphingosineamidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variantthereof, more preferably from a mammalian ribosomal protein Large 32gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomalprotein Large 21 gene (RPL21), a mammalian ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle(ATP5A1) gene, a mammalian hydroxysteroid (17-beta) dehydrogenase 4 gene(HSD17B4), a mammalian androgen-induced 1 gene (AIG1), a mammaliancyto-chrome c oxidase subunit VIc gene (COX6C), or a mammalianN-acylsphingosine ami-dohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, most preferably from a human ribosomal proteinLarge 32 gene (RPL32), a human ribosomal protein Large 35 gene (RPL35),a human ribosomal protein Large 21 gene (RPL21), a human ATP syn-thase,H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiacmuscle (ATP5A1) gene, a human hydroxysteroid (17-beta) dehydrogenase 4gene (HSD17B4), a human androgen-induced 1 gene (AIG1), a humancytochrome c oxidase subunit VIc gene (COX6C), or a humanN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, wherein preferably the 5′-UTR does not comprisethe 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′-UTRcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID NO. 1368, or SEQ IDNOs 1412-1420 of the patent application WO2013/143700, or acorresponding RNA sequence, or wherein the at least one 5′-UTR comprisesor consists of a fragment of a nucleic acid sequence, which has anidentity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID NO.1368, or SEQ ID NOs 1412-1420 of the patent application WO2013/143700,wherein, preferably, the fragment is as described above, i.e. being acontinuous stretch of nucleotides representing at least 20% etc. of thefull-length 5′-UTR. Preferably, the fragment exhibits a length of atleast about 20 nucleotides or more, preferably of at least about 30nucleotides or more, more preferably of at least about 40 nucleotides ormore. Preferably, the fragment is a functional fragment as describedherein.

Accordingly, in a particularly preferred embodiment, the 5′-UTRcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according SEQ ID NO. 1414 of the patentapplication WO2013/143700 (5′-UTR of ATP5A1 lacking the 5′ terminaloligopyrimidine tract) or preferably to a corresponding RNA sequence, orwherein the at least one 5′-UTR comprises or consists of a fragment of anucleic acid sequence, which has an identity of at least about 40%,preferably of at least about 50%, preferably of at least about 60%,preferably of at least about 70%, more preferably of at least about 80%,more preferably of at least about 90%, even more preferably of at leastabout 95%, even more preferably of at least about 99% to the nucleicacid sequence according to SEQ ID NO. 1414 of the patent applicationWO2013/143700 or more preferably to a corresponding RNA sequence,wherein, preferably, the fragment is as described above, i.e. being acontinuous stretch of nucleotides representing at least 20% etc. of thefull-length 5′-UTR. Preferably, the fragment exhibits a length of atleast about 20 nucleotides or more, preferably of at least about 30nucleotides or more, more preferably of at least about 40 nucleotides ormore. Preferably, the fragment is a functional fragment as describedherein.

In a further preferred embodiment, the nucleic acid molecule of theinventive polymeric carrier cargo complex as defined herein and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex, preferably an mRNA, comprises at leastone 3′-UTR.

More preferably, the mRNA comprises or consists of a nucleic acidsequence derived from the 3′-UTR of a chordate gene, preferably avertebrate gene, more preferably a mammalian gene, most preferably ahuman gene, or from a variant of the 3′-UTR of a chordate gene,preferably a vertebrate gene, more preferably a mammalian gene, mostpreferably a human gene.

Preferably, the inventive mRNA sequence comprises a 3′-UTR, which may bederivable from a gene that relates to an mRNA with an enhanced half-life(that provides a stable mRNA), for example a 3′-UTR as defined anddescribed below.

In a particularly preferred embodiment, the 3′-UTR comprises or consistsof a nucleic acid sequence, which is derived from a 3′-UTR of a geneselected from the group consisting of an albumin gene, an α-globin gene,a 3-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and acollagen alpha gene, such as a collagen alpha 1(I) gene, or from avariant of a 3′-UTR of a gene selected from the group consisting of analbumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylasegene, a lipoxygenase gene, and a collagen alpha gene, such as a collagenalpha 1(I) gene according to SEQ ID NO. 1369-1390 of the patentapplication WO2013/143700 whose disclosure is incorporated herein byreference. In a particularly preferred embodiment, the 3′-UTR comprisesor consists of a nucleic acid sequence, which is derived from a 3′-UTRof an albumin gene, preferably a vertebrate albumin gene, morepreferably a mammalian albumin gene, most preferably a human albumingene according SEQ ID No: 1369 of the patent application WO2013/143700.The mRNA sequence may comprise or consist of a nucleic acid sequence,which is derived from the 3′-UTR of the human albumin gene according toGenBank Accession number NM_000477.5, or from a fragment or variantthereof.

In this context, it is particularly preferred that the mRNA comprises a3′-UTR comprising a corresponding RNA sequence derived from the nucleicacid sequences according to SEQ ID NO. 1369-1390 of the patentapplication WO2013/143700 or a fragment, homolog or variant thereof.

Most preferably the 3′-UTR comprises the nucleic acid sequence derivedfrom a fragment of the human albumin gene according to SEQ ID No: 1376of the patent application WO2013/143700, in the following referred to asSEQ ID NO. 33.

Nucleotide sequence of 3′-UTR element of human albumin gene(SEQ ID NO. 387) CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCT

In another particularly preferred embodiment, the 3′-UTR comprises orconsists of a nucleic acid sequence, which is derived from a 3′-UTR ofan α-globin gene, preferably a vertebrate α- or β-globin gene, morepreferably a mammalian α- or β-globin gene, most preferably a human α-or β-globin gene according to SEQ ID NO. 1370 of the patent applicationWO2013/143700 (3′-UTR of Homo sapiens hemoglobin, alpha 1 (HBA1)), oraccording to SEQ ID NO. 1371 of the patent application WO2013/143700(3′-UTR of Homo sapiens hemoglobin, alpha 2 (HBA2)), or according to SEQID NO. 1372 of the patent application WO2013/143700 (3′-UTR of Homosapiens hemoglobin, beta (HBB)).

For example, the 3′-UTR may comprise or consist of the center,α-complex-binding portion of the 3′-UTR of an α-globin gene, such as ofa human α-globin gene, preferably according to SEQ ID NO. 388(corresponding to SEQ ID NO. 1393 of the patent applicationWO2013/143700).

Nucleotide sequence of 3′ UTR element of an α-globin gene(SEQ ID NO. 388) GCCCGATGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCG

In this context it is particularly preferred that the 3′-UTR of theinventive mRNA comprises or consists of a corresponding RNA sequence ofthe nucleic acid sequence according to the above or a homolog, afragment or variant thereof.

The term ‘a nucleic acid sequence, which is derived from the 3′-UTR of a[ . . . ] gene’ preferably refers to a nucleic acid sequence, which isbased on the 3′-UTR sequence of a [ . . . ] gene or on a part thereof,such as on the 3′-UTR of an albumin gene, an α-globin gene, a β-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagenalpha gene, such as a collagen alpha 1(I) gene, preferably of an albumingene or on a part thereof. This term includes sequences corresponding tothe entire 3′-UTR sequence, i.e. the full length 3′-UTR sequence of agene, and sequences corresponding to a fragment of the 3′-UTR sequenceof a gene, such as an albumin gene, α-globin gene, β-globin gene,tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene,such as a collagen alpha 1(I) gene, preferably of an albumin gene.

The term ‘a nucleic acid sequence, which is derived from a variant ofthe 3′-UTR of a [ . . . ] gene’ preferably refers to a nucleic acidsequence, which is based on a variant of the 3′-UTR sequence of a gene,such as on a variant of the 3′-UTR of an albumin gene, an α-globin gene,a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or acollagen alpha gene, such as a collagen alpha 1(I) gene, or on a partthereof as described above. This term includes sequences correspondingto the entire sequence of the variant of the 3′-UTR of a gene, i.e. thefull length variant 3′-UTR sequence of a gene, and sequencescorresponding to a fragment of the variant 3′-UTR sequence of a gene. Afragment in this context preferably consists of a continuous stretch ofnucleotides corresponding to a continuous stretch of nucleotides in thefull-length variant 3′-UTR, which represents at least 20%, preferably atleast 30%, more preferably at least 40%, more preferably at least 50%,even more preferably at least 60%, even more preferably at least 70%,even more preferably at least 80%, and most preferably at least 90% ofthe full-length variant 3′-UTR. Such a fragment of a variant, in thesense of the present invention, is preferably a functional fragment of avariant as described herein.

Preferably, the at least one 5′-UTR and the at least one 3′-UTR actsynergistically to increase protein production from the nucleic acidmolecule of the inventive polymeric carrier cargo complex as definedherein and/or the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex as described above.

In a particularly preferred embodiment, the nucleic acid molecule of theinventive polymeric carrier cargo complex as defined herein and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex comprises a histone stem-loopsequence/structure. Such histone stem-loop sequences are preferablyselected from histone stem-loop sequences as disclosed in WO2012/019780, whose disclosure is incorporated herewith by reference.

A histone stem-loop sequence, suitable to be used within the presentinvention, is preferably selected from at least one of the followingformulae (VI) or (VII):

Formula (VI) (Stem-Loop Sequence without Stem Bordering Elements):

Formula (VII) (Stem-Loop Sequence with Stem Bordering Elements):

wherein:

-   stem1 or stem2 bordering elements N₁₋₆ is a consecutive sequence of    1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more    preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each    N is independently from another selected from a nucleotide selected    from A, U, T, G and C, or a nucleotide analogue thereof;-   stem1 [N₀₋₂ GN₃₋₅] is reverse complementary or partially reverse    complementary with element stem2, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof, and    -   wherein G is guanosine or an analogue thereof, and may be        optionally replaced by a cytidine or an analogue thereof,        provided that its complementary nucleotide cytidine in stem2 is        replaced by guanosine;-   loop sequence [N₀₋₄ (U/T)N₀₋₄] is located between elements stem1 and    stem2, and is a consecutive sequence of 3 to 5 nucleotides, more    preferably of 4 nucleotides;    -   wherein each No-4 is independent from another a consecutive        sequence of 0 to 4, preferably of 1 to 3, more preferably of 1        to 2 N, wherein each N is independently from another selected        from a nucleotide selected from A, U, T, G and C or a nucleotide        analogue thereof; and    -   wherein U/T represents uridine, or optionally thymidine;-   stem2 [N₃₋₅ CN₀₋₂] is reverse complementary or partially reverse    complementary with element stem1, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        or C or a nucleotide analogue thereof; and    -   wherein C is cytidine or an analogue thereof, and may be        optionally replaced by a guanosine or an analogue thereof        provided that its complementary nucleoside guanosine in stem1 is        replaced by cytidine;        wherein        stem1 and stem2 are capable of base pairing with each other        forming a reverse complementary sequence, wherein base pairing        may occur between stem1 and stem2, e.g. by Watson-Crick base        pairing of nucleotides A and U/T or G and C or by        non-Watson-Crick base pairing e.g. wobble base pairing, reverse        Watson-Crick base pairing, Hoogsteen base pairing, reverse        Hoogsteen base pairing or are capable of base pairing with each        other forming a partially reverse complementary sequence,        wherein an incomplete base pairing may occur between stem1 and        stem2, on the basis that one ore more bases in one stem do not        have a complementary base in the reverse complementary sequence        of the other stem.

According to a further preferred embodiment of the first inventiveaspect, the nucleic acid molecule of the inventive polymeric carriercargo complex as defined herein and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex maycomprise at least one histone stem-loop sequence according to at leastone of the following specific formulae (VIa) or (VIIa):

Formula (VIa) (Stem-Loop Sequence without Stem Bordering Elements):

Formula (VIIa) (Stem-Loop Sequence with Stem Bordering Elements):

wherein:N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of thefirst aspect, the nucleic acid molecule of the inventive polymericcarrier cargo complex as defined herein and/or the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex may comprise at least one histone stem-loop sequence accordingto at least one of the following specific formulae (VIb) or (VIIb):

Formula (VIb) (Stem-Loop Sequence without Stem Bordering Elements):

Formula (VIIb) (Stem-Loop Sequence with Stem Bordering Elements):

wherein:N, C, G, T and U are as defined above.

A particular preferred histone stem-loop sequence is the nucleic acidsequence according to SEQ ID NO. 389.

Histone stem-loop nucleotide sequence (SEQ ID NO. 389)CAAAGGCTCTTTTCAGAGCCACCA

More preferably the stem-loop sequence is the corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID NO. 390.

Histone stem-loop RNA sequence (SEQ ID NO. 390) CAAAGGCUCUUUUCAGAGCCACCA

In a particularly preferred embodiment, the nucleic acid molecule of theinventive polymeric carrier cargo complex as defined herein and/or thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex comprises a nucleic acid sequencederived from a 5′-TOP-UTR, a GC-optimized coding sequence, a nucleicacid sequence derived from the 3′-UTR of an albumin gene, apoly(A)-sequence, a poly(C)-sequence, and a histone stem loop. It isparticularly preferred that the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex isan mRNA molecule, which comprises a nucleic acid sequence derived from a5′-TOP-UTR, a GC-optimized coding sequence, a nucleic acid sequencederived from the 3′-UTR of an albumin gene, a poly(A)-sequence, apoly(C)-sequence, and a histone stem loop, wherein each of thesefeatures is preferably as defined herein.

Nevertheless, substitutions, additions or eliminations of bases arepreferably carried out with the nucleic acid molecule of the inventivepolymeric carrier cargo complex as defined herein and/or the secondnucleic acid molecule administered in combination with the polymericcarrier cargo complex, especially if the nucleic acid is in the form ofan mRNA, using a DNA matrix for preparation of the nucleic acid moleculeby techniques of the well known site directed mutagenesis or with anoligonucleotide ligation strategy (see e.g. Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rded., Cold Spring Harbor, N.Y., 2001). In such a process, for preparationof the nucleic acid molecule of the inventive polymeric carrier cargocomplex as defined herein and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex,especially if the nucleic acid is in the form of an mRNA, acorresponding DNA molecule may be transcribed in vitro. This DNA matrixpreferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, forin vitro transcription, which is followed by the desired nucleotidesequence for the nucleic acid molecule, e.g. mRNA, to be prepared and atermination signal for in vitro transcription. The DNA molecule, whichforms the matrix of the at least one RNA of interest, may be prepared byfermentative proliferation and subsequent isolation as part of a plasmidwhich can be replicated in bacteria. Plasmids which may be mentioned assuitable for the present invention are e.g. the plasmids pT7Ts (GenBankaccession number U26404; Lai et al., Development 1995, 121: 2349 to2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; fromPromega) and pSP64 (GenBank accession number X65327); cf. also Mezei andStorts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCRTechnology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

Nucleic acid molecules used according to the invention as defined hereinmay be prepared using any method known in the art, including syntheticmethods such as e.g. solid phase synthesis, as well as in vitro methods,such as in vitro transcription reactions.

According to another particularly preferred embodiment, the nucleic acidmolecule of the inventive polymeric carrier cargo complex as definedherein and/or the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex, especially if thenucleic acid is in the form of a coding nucleic acid molecule mayadditionally or alternatively encode a secretory signal peptide. Suchsignal peptides are sequences, which typically exhibit a length of about15 to 30 amino acids and are preferably located at the N-terminus of theencoded peptide, without being limited thereto. Signal peptides asdefined herein preferably allow the transport of the protein or peptideas encoded by the nucleic acid molecule of the inventive polymericcarrier cargo complex as defined herein and/or the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex, especially if the nucleic acid is in the form of an mRNA, intoa defined cellular compartment, preferably the cell surface, theendoplasmic reticulum (ER) or the endosomal-lysosomal compartment.Examples of secretory signal peptide sequences as defined hereininclude, without being limited thereto, signal sequences of classical ornon-classical MHC-molecules (e.g. signal sequences of MHC I and IImolecules, e.g. of the MHC class I molecule HLA-A*0201), signalsequences of cytokines or immunoglobulins as defined herein, signalsequences of the invariant chain of immunoglobulins or antibodies asdefined herein, signal sequences of Lamp1, Tapasin, Erp57, Calreticulin,Calnexin, and further membrane associated proteins or of proteinsassociated with the endoplasmic reticulum (ER) or theendosomal-lysosomal compartment. Particularly preferably, signalsequences of MHC class I molecule HLA-A*0201 may be used according tothe present invention.

Any of the above modifications may be applied to the nucleic acidmolecule of the inventive polymeric carrier cargo complex as definedherein, or to the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex and/or to anynucleic acid as used in the context of the present invention and may be,if suitable or necessary, be combined with each other in anycombination, provided, these combinations of modifications do notinterfere with each other in the respective nucleic acid. A personskilled in the art will be able to take his choice accordingly.

The nucleic acid molecule of the inventive polymeric carrier cargocomplex as defined herein and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex aswell as proteins or peptides as encoded by these nucleic acid moleculesmay comprise fragments or variants of those sequences. Such fragments orvariants may typically comprise a sequence having a sequence identitywith one of the above mentioned nucleic acids, or with one of theproteins or peptides or sequences, if encoded by the at least onenucleic acid molecule, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,preferably at least 70%, more preferably at least 80%, equally morepreferably at least 85%, even more preferably at least 90% and mostpreferably at least 95% or even 97%, 98% or 99%, to the entire wild typesequence, either on nucleic acid level or on amino acid level.

“Fragments” of proteins or peptides in the context of the presentinvention (encoded by a nucleic acid as defined herein) may comprise asequence of a protein or peptide as defined herein, which is, withregard to its amino acid sequence (or its encoded nucleic acidmolecule), N-terminally, C-terminally and/or intrasequentially truncatedcompared to the amino acid sequence of the original (native) protein (orits encoded nucleic acid molecule). Such truncation may thus occureither on the amino acid level or correspondingly on the nucleic acidlevel. A sequence identity with respect to such a fragment as definedherein may therefore preferably refer to the entire protein or peptideas defined herein or to the entire (coding) nucleic acid molecule ofsuch a protein or peptide. Likewise, “fragments” of nucleic acids in thecontext of the present invention may comprise a sequence of a nucleicacid as defined herein, which is, with regard to its nucleic acidmolecule 5′-, 3′- and/or intrasequentially truncated compared to thenucleic acid molecule of the original (native) nucleic acid molecule. Asequence identity with respect to such a fragment as defined herein maytherefore preferably refer to the entire nucleic acid as defined herein.

Fragments of proteins or peptides in the context of the presentinvention (e.g. as encoded by the nucleic acid molecule of the inventivepolymeric carrier cargo complex and/or by the second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex) may furthermore comprise a sequence of a protein or peptide asdefined herein, which has a length of about 6 to about 20 or even moreamino acids, e.g. fragments as processed and presented by MHC class Imolecules, preferably having a length of about 8 to about 10 aminoacids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino acids), orfragments as processed and presented by MHC class II molecules,preferably having a length of about 13 or more amino acids, e.g. 13, 14,15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragmentsmay be selected from any part of the amino acid sequence. Thesefragments are typically recognized by T-cells in form of a complexconsisting of the peptide fragment and an MHC molecule, i.e. thefragments are typically not recognized in their native form.

Fragments of proteins or peptides as defined herein (e.g. as encoded bythe nucleic acid molecule of the inventive polymeric carrier cargocomplex and/or by the second nucleic acid molecule administered incombination with the polymeric carrier cargo complex) may also compriseepitopes of those proteins or peptides. Epitopes (also called “antigendeterminants”) in the context of the present invention are typicallyfragments located on the outer surface of (native) proteins or peptidesas defined herein, preferably having 5 to 15 amino acids, morepreferably having 5 to 12 amino acids, even more preferably having 6 to9 amino acids, which may be recognized by antibodies or B-cellreceptors, i.e. in their native form. Such epitopes of proteins orpeptides may furthermore be selected from any of the herein mentionedvariants of such proteins or peptides. In this context antigenicdeterminants can be conformational or discontinous epitopes which arecomposed of segments of the proteins or peptides as defined herein thatare discontinuous in the amino acid sequence of the proteins or peptidesas defined herein but are brought together in the three-dimensionalstructure or continuous or linear epitopes which are composed of asingle polypeptide chain.

“Variants” of proteins or peptides as defined in the context of thepresent invention (e.g. as encoded by a nucleic acid as defined herein)may be encoded by the nucleic acid molecule of the inventive polymericcarrier cargo complex and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex.Thereby, a protein or peptide may be generated, having an amino acidsequence which differs from the original sequence in one or moremutation(s), such as one or more substituted, inserted and/or deletedamino acid(s). Preferably, these fragments and/or variants have the samebiological function or specific activity compared to the full-lengthnative protein, e.g. its specific antigenic property.

“Variants” of proteins or peptides as defined in the context of thepresent invention (e.g. as encoded by a nucleic acid as defined herein)may comprise conservative amino acid substitution(s) compared to theirnative, i.e. non-mutated physiological, sequence. Those amino acidsequences as well as their encoding nucleotide sequences in particularfall under the term variants as defined herein. Substitutions in whichamino acids, which originate from the same class, are exchanged for oneanother are called conservative substitutions. In particular, these areamino acids having aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or amino acids,the side chains of which can enter into hydrogen bridges, e.g. sidechains, which have a hydroxyl function. This means that e.g. an aminoacid having a polar side chain is replaced by another amino acid havinga likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions, which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam).

Furthermore, variants of proteins or peptides as defined herein, whichmay be encoded by the nucleic acid molecule of the inventive polymericcarrier cargo complex and/or the second nucleic acid moleculeadministered in combination with the polymeric carrier cargo complex,may also comprise those sequences, wherein nucleotides of the nucleicacid are exchanged according to the degeneration of the genetic code,without leading to an alteration of the respective amino acid sequenceof the protein or peptide, i.e. the amino acid sequence or at least partthereof may not differ from the original sequence in one or moremutation(s) within the above meaning.

In order to determine the percentage, to which two sequences areidentical, e.g. nucleic acid sequences or amino acid sequences asdefined herein, preferably the amino acid sequences encoded by a nucleicacid sequence of the polymeric carrier as defined herein or the aminoacid sequences themselves, the sequences can be aligned in order to besubsequently compared to one another. Therefore, e.g. a position of afirst sequence may be compared with the corresponding position of thesecond sequence. If a position in the first sequence is occupied by thesame component as is the case at a position in the second sequence, thetwo sequences are identical at this position. If this is not the case,the sequences differ at this position. If insertions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe first sequence to allow a further alignment. If deletions occur inthe second sequence in comparison to the first sequence, gaps can beinserted into the second sequence to allow a further alignment. Thepercentage to which two sequences are identical is then a function ofthe number of identical positions divided by the total number ofpositions including those positions, which are only occupied in onesequence. The percentage to which two sequences are identical can bedetermined using a mathematical algorithm. A preferred, but notlimiting, example of a mathematical algorithm which can be used is thealgorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul etal. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm isintegrated in the BLAST program. Sequences, which are identical to thesequences of the present invention to a certain extent, can beidentified by this program.

In the inventive polymeric carrier cargo complex, the cationic componentof the polymeric carrier as defined herein and the nucleic acid cargoare typically provided in a molar ratio of about 1 to 10000, preferablyin a molar ratio of about 5 to 5000, more preferably in a molar ratio ofabout 10 to 2500, even more preferably in a molar ratio of about 25 to2000, and most preferably in a molar ratio of about 25 to 1000 ofpolymeric carrier to nucleic acid.

Furthermore, in the inventive polymeric carrier cargo complex, thecationic component of the polymeric carrier as defined herein and thenucleic acid cargo are preferably provided in an N/P-ratio of at least0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5 or 2. Preferably, the N/P-ratiolies within a range of about 0.1, 0.3, 0.4, 0.5, 0.75, 1.0, 1.5 or 2 to20, preferably in a range of about 0.2 (0.5 or 0.75 or 1.0) to 12, andeven more preferably in an N/P-ratio of about 0.4 (0.75 or 1.0) to 10.Most preferably, the N/P ratio lies in a ratio between 0.1 and 0.9. Inthis context, the N/P ratio is a measure of the ionic charge of thecationic (side chain) component of the polymeric carrier or of thepolymeric carrier as such. In particular, if the cationic properties ofthe cationic component are generated by nitrogens (of the amino acidside chains), the N/P ratio expresses the ratio of basic nitrogen atomsto phosphate residues in the nucleotide backbone, considering that (sidechain) nitrogen atoms in the cationic component of the polymeric carriercontribute to positive charges and phosphate of the phosphate backboneof the nucleic acid contribute to the negative charge. A formula isgiven in the Examples. The N/P-ratio is defined as thenitrogen/phosphate ratio (N/P-ratio) of the entire inventive polymericcarrier cargo complex. This is typically illustrative for thecontent/amount of cationic components, in the polymeric carrier andcharacteristic for the content/amount of nucleic acids bound orcomplexed in the inventive polymeric carrier cargo complex. It may becalculated on the basis that, for example, 1 μg RNA typically containsabout 3 nmol phosphate residues, provided that RNA exhibits astatistical distribution of bases. Additionally, 1 nmol peptidetypically contains about x nmol nitrogen residues, dependent on themolecular weight and the number of its (cationic) amino acids.

In this context it is preferable that in the inventive polymeric carriercargo complex, the cationic component of the polymeric carrier asdefined herein and the nucleic acid cargo are provided in an N/P-ratioof at least about 1 or, preferably, of a range of about 1 to 20 for invitro transfection purposes.

If the expression of an encoded protein or the transcription of anencoded nucleic acid e.g. an mRNA or siRNA of the nucleic acid cargo isintended for therapeutical purposes (in vivo application) an N/P ratioof at least 0.1 (0.2, 0.3, 0.4, 0.5, 0.6), preferably of a range ofabout 0.1 (0.2, 0.3, 0.4, 0.5, or 0.6) to 1.5 is preferred. Even morepreferred is an N/P ratio range of 0.2 to 0.9 or an N/P ratio range of0.5 to 0.9. In the case that the inventive polymeric carrier cargocomplex is used for (in vivo) immunostimulation e.g. as animmunostimulating agent or adjuvant (for the purpose to induce an innateimmune response), an N/P ratio of about 0.1 to 20 is preferred, moreparticular an N/P ratio of 0.1 to 5 or 0.1 to 1.5.

In the specific case that the induction of IFN-α is intended using theinventive polymeric cargo complex as an (in vivo) immunostimulatingagent or adjuvant an N/P ratio of at least 0.1 (0.2, 0.3, 0.4, 0.5, or0.6) or an N/P ratio range of 0.1 to 1 is preferred or more preferred isan N/P ratio range of 0.2 to 0.9 or an N/P ratio range of 0.5 to 0.9.Otherwise if the induction of TNFα is intended using the inventivepolymeric cargo complex as an (in vivo) immunostimulating agent oradjuvant an N/P ratio of 1 to 20 is particularly preferred.

The N/P ratio significantly influences the surface charge of theresulting inventive polymeric carrier cargo complex. Thus it ispreferable that the resulting inventive polymeric carrier cargo complexis positively charged for in vitro transfection purposes and negativelyor neutrally charged for in vivo transfection purposes, especially ifthe expression of an encoded protein or the transcription of an encodednucleic acid of the nucleic acid cargo is intended. The surface chargeof the resulting inventive polymeric carrier cargo complex can beindicated as Zetapotential which may be measured by Dopplerelectrophoresis method using a Zetasizer Nano (Malvern Instruments,Malvern, UK).

The molar ratio of the nucleic acid molecule used as a cargo in thepolymeric carrier cargo complex (“first nucleic acid molecule”) to thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex is preferably in the range from 0.01 to100, more preferably in the range from 0.1 to 10, even more preferablyin the range from 0.5 to 2, most preferably about 1.

The second nucleic acid molecule, which is administered in combinationwith the polymeric carrier cargo complex, is typically used innon-packaged form, i.e. the second nucleic acid molecule, preferably anRNA, in the context of the present invention is preferably not packagedin particles. Preferably, the second nucleic acid molecule, which isadministered in combination with the polymeric carrier cargo complex, isnot packaged in a virus particle, an inactivated virus particle or avirus-like particle. “Non-packaged” in this context refers to a nucleicacid molecule, which may be a naked nucleic acid molecule or a nucleicacid molecule, which is complexed by another compound, preferably acationic compound. In one embodiment, the second nucleic acid molecule,preferably an RNA, is complexed by another compound, thus forminganother polymeric complex distinct from the polymeric carrier cargocomplex as defined herein. Accordingly, the second nucleic acid moleculemay be in the form of a complex, wherein the complex comprising thesecond nucleic acid molecule is distinct from the polymeric carriercargo complex, in particular with respect to the nucleic acid sequenceof the respective first nucleic acid molecules and/or with respect tothe compound, by which the first nucleic acid molecule, respectively, iscomplexed.

Accordingly, in a preferred embodiment, the second nucleic acidmolecule, preferably an RNA, encoding a peptide or a protein, isadministered in the form of a naked nucleic acid. Alternatively, thesecond nucleic acid molecule, preferably an RNA, is complexed by acationic or polycationic compound. According to one embodiment, thecationic or polycationic compound is selected from protamine,nucleoline, spermine or spermidine, poly-L-lysine (PLL), basicpolypeptides, poly-arginine, cell penetrating peptides (CPPs), chimericCPPs, including Transportan, or MPG peptides, HIV-binding peptides, Tat,HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of thepenetratin family, including Penetratin, Antennapedia-derived peptides(from Drosophila antennapedia), pAntp, pIs1, antimicrobial-derived CPPsincluding Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derivedpeptides, SAP, MAP, KALA, PpTG20, Proline-rich peptides, L-oligomers,Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin,poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides,HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domainsincluding PTDs, PpT620, prolin-rich peptides, arginine-rich peptides,lysine-rich peptides, Pep-1, and Calcitonin peptide(s), or from proteinsor peptides having the following total formula:(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), wherein1+m+n+o+x=8-15, and 1, m, n or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8 provided, that theoverall content of Xaa does not exceed 50% of all amino acids of theoligopeptide,

or from oligoarginines including Arg₇, Arg₈, Arg₉, Arg₇, H₃R₉, R₉H₃,H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R,or from cationic polysaccharides, including chitosan, polybrene,cationic polymers, polyethyleneimine (PEI), cationic lipids, DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE(Dioleyl phosphatidylethanol-amine), DOSPA, DODAB, DOIC, DMEPC, DOGS(Dioctadecylamidoglicylspermin), DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(a-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or from cationic or polycationic polymers, includingmodified polyaminoacids, including as (3-aminoacid-polymers or reversedpolyamides, modified polyethylenes, including PVP(poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, includingpDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified Amidoaminesincluding pAMAM (poly(amidoamine)), modified polybetaaminoester (PBAE),including diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, dendrimers, includingpolypropylamine dendrimers or pAMAM based dendrimers, polyimine(s),including PEI: poly(ethyleneimine), poly(propyleneimine),polyallylamine, sugar backbone based polymers, including cyclodextrinbased polymers, dextran based polymers, Chitosan, silan backbone basedpolymers, including PMOXA-PDMS copolymers, blockpolymers consisting of acombination of one or more cationic blocks (selected of a cationicpolymer as defined above) and of one or more hydrophilic- or hydrophobicblocks (including polyethyleneglycol).

Preferably, the second nucleic acid molecule is administered incombination with the polymeric carrier cargo complex, without beingcomprised in the polymeric carrier cargo complex. In particular, thesecond nucleic acid molecule is administered in combination with thepolymeric carrier cargo complex as defined herein, without physicallybeing a part or component of the polymeric carrier cargo complex.Preferably, the second nucleic acid molecule is not bound (e.g.covalently) to the polymeric carrier cargo complex. Further preferably,the at least one first nucleic acid molecule of the inventive polymericcarrier cargo complex and the at least one second nucleic acid molecule,which is administered together with the polymeric carrier cargo complex,are not complexed by the same polymeric carrier.

In a further aspect the present invention also provides a method ofpreparing the inventive polymeric carrier cargo complex as definedherein comprising following steps:

-   a) providing at least one cationic protein or peptide as defined    herein and/or at least one cationic or polycationic polymer and    optionally at least one amino acid component (AA) as defined herein,    each comprising at least one —SH moiety,-   b) providing at least one first nucleic acid molecule as defined    herein, preferably in the above mentioned ratios,-   c) mixing the components provided in steps a) and b), preferably in    a basic or neutral milieu as defined herein, preferably in the    presence of oxygen or a further starter as defined herein,    preferably at a pH, at a temperature and at time as defined herein,    and thereby condensing and thus polymerizing the cationic components    provided in step a) with each other via disulfide bonds (in a    condensation polymerization or polycondensation) to obtain the    polymeric carrier and complexing the nucleic acid molecule provided    in step b) with the cationic components provided in step a)-   d) optionally purifying the inventive polymeric carrier cargo    complex obtained according to step c), preferably using a method as    defined herein;-   e) optionally lyophilization of the inventive polymeric carrier    cargo complex obtained according to step c) or d).

The method of preparing the inventive polymeric carrier cargo complex asdefined herein comprises a multi-step condensation polymerization orpolycondensation reaction via —SH moieties of the educts e.g. cationicpeptides or polymers as defined herein and optionally further amino acidcomponents (AA) in step c). The condensation polymerization orpolycondensation reaction which occurs simultaneously to thecomplexation or electrostratic binding of the nucleic acid moleculepreferably leads to the inventive polymeric carrier cargo complexwherein the polymeric carrier is a condensation polymer, wherein thesingle components are linked by disulfide bonds.

As defined herein in a step a) of the inventive method of preparing theinventive polymeric carrier cargo complex, at least one cationic orpolycationic protein or peptide as defined herein and/or at least onecationic or polycationic polymer as defined herein are provided,preferably in the ratios indicated above. These components are mixed instep c) with the nucleic acid molecule provided in step b), preferablyin a basic or neutral milieu as defined herein, preferably in thepresence of oxygen or a further starter as defined herein, preferably ata pH, and at a temperature and at a time as defined herein, and therebycondensing and thus polymerizing these components with each other viadisulfide bonds (in a condensation polymerization or polycondensation)to obtain a polymeric carrier complexed to the at least one firstnucleic acid molecule as defined herein.

According to an alternative, in step a) of the inventive method ofpreparing the inventive polymeric carrier cargo complex at least onecationic or polycationic protein or peptide and/or at least one cationicor polycationic polymer are provided as defined herein, and optionallyat least one amino acid component (AA), are provided in step a) asdefined herein, and are used for a condensation polymerization orpolycondensation and complexation reaction prior to adding the nucleicacid of step b) but using the same polymerization conditions outlinedfor step c). The polymerized polymeric carrier and the nucleic acid ofstep b) are then mixed in step c). Preferably, the components are allprovided in the ratios indicated above and mixed, preferably in a basicor neutral milieu as defined herein, preferably in the presence ofoxygen or a further starter as defined herein, preferably at a pH, at atemperature and at time as defined herein. Upon mixing and starting thereaction, the components are condensed and thus polymerized with eachother via disulfide bonds (in a condensation polymerization orpolycondensation) to obtain a polymeric carrier complexed to the nucleicacid molecule as defined herein.

In both of the above alternatives, different polymeric carriers,particularly different peptides and/or different polymers, and may beselected in the condensation polymerization as indicated above. In thiscontext, the selection of different component(s) of the polymericcarrier is typically dependent upon the desired properties of the finalpolymeric carrier and the desired cationic strength of the finalpolymeric carrier. Accordingly, the content of cationic components, mayfurthermore be “diluted” or modified in the above alternative of step a)e.g. by introducing an amino acid component (AA) as defined herein,preferably in the above defined ratios. Thereby, a modified polymericcarrier may be obtained, wherein the cationic character of theunmodified polymeric carrier typically remains in the limitations asdefined herein. The properties of the final polymeric carrier may thusbe adjusted as desired with properties of components (AA) by insertingamino acid component (AA) as defined herein in steps a).

In step c), the at least one cationic or polycationic protein or peptideas defined herein and/or at least one cationic or polycationic polymeras defined herein, and optionally at least one amino acid component (AA)and the at least one first nucleic acid as defined herein, arepreferably contained in a basic or neutral milieu in the step a) of theinventive method of preparing the inventive polymeric carrier cargocomplex. Such a basic or neutral milieu typically exhibits a pH range ofabout 5 to about 10, preferably a pH range of about 6 to about 9, morepreferably a pH range of about 7 to about 8, e.g. about 6.5, 7, 7.5, 8,8.5, or 9 or any range selected from any two of these or theaforementioned values.

Furthermore, the temperature of the solution in step c) is preferably ina range of about 5° C. to about 60° C., more preferably in a range ofabout 15° C. to about 40° C., even more preferably in a range of about20° C. to about 30° C., and most preferably in a range of about 20° C.to about 25° C., e.g. about 25° C.

In step c) of the inventive method of preparing the inventive polymericcarrier cargo complex as defined herein buffers may be used as suitable.Preferred buffers may comprise, but are not limited to carbonatebuffers, borate buffers, Bicine buffer, CHES buffer, CAPS buffer,Ethanolamine containing buffers, HEPES, MOPS buffer, Phosphate buffer,PIPES buffer, Tris buffer, Tricine buffer, TAPS buffer, and/or TESbuffer as buffering agents. Particularly preferred is a carbonatebuffer.

Upon mixing the components, preferably in the presence of oxygen,preferably in the presence of a basic or neutral mileu as definedherein, the condensation polymerization or polycondensation reaction andthe complexation of the at least one nucleic acid molecule is started.For this purpose, the mixture in step c) is preferably exposed to oxygenor may be started using a further starter, e.g. a catalytic amount of anoxidizing agent, e.g. DMSO, etc. Upon start of the condensationpolymerization or polycondensation reaction of the at least one cationicor polycationic protein or peptide and/or at least one cationic orpolycationic polymer and optionally at least one amino acid component(AA) as defined herein, are condensed and thus polymerized with eachother via disulfide bonds (condensation polymerization orpolycondensation). In this reaction step a) preferably linear polymersare created using monomers with at least one reactive —SH moiety, i.e.at least one cationic or polycationic protein or peptide and/or at leastone cationic or polycationic polymer and optionally at least one aminoacid component (AA) as defined herein, each component exhibiting atleast one free —SH-moieties as defined herein, e.g. at their terminalends. However, components with more than one, preferably two free—SH-moieties may be used, which may lead to branched polymers.Simultaneously to the polymerization reaction the cationic polymers bindto the at least one nucleic acid molecule and thereby complexing it.

According to one alternative, the inventive polymeric carrier cargocomplex additionally may be modified with a component (AA) as definedherein.

According to a first example, a component (AA) (e.g. a ligand) isattached to the cationic component prior to providing the cationiccomponent in step a) via any functionality as defined herein, e.g. a —SHmoiety. This component (AA) or (e.g. a ligand) is preferably attached tothe cationic component at one terminus of these components. If theattachment is carried out via —SH bonds, the cationic components arepreferably provided with two (or even more) —SH-moieties. The component(AA) or (e.g. a ligand) preferably carries only one —SH moiety. In thiscase, one —SH moiety of the cationic component is preferably protectedin a first step using a protecting group as known in the art. Then, thecationic component may be bound to a component L to form a firstdisulfide bond via the non-protected —SH moiety. The protected—SH-moiety of the cationic component is then typically deprotected forfurther reactions.

Alternatively, the above mentioned component (AA) or (e.g. a ligand) maybe used in step c) to be coupled with the cationic components providedin step a) above, e.g. via disulfide bonds without blocking the free —SHmoieties. But in this context all methods known to a skilled person ordefined herein may be used to attach the component (AA) to the cationiccomponent or to the polymeric carrier.

Alternatively, a component (AA) or (e.g. a ligand) can be bound to theinventive polymeric carrier cargo complex after step c) via anyfunctionality as defined herein, e.g. a —SH moiety. In this context itis preferable that the component (AA) (e.g. a ligand) is bound via free—SH moieties of the polymeric carrier components.

According to step c) of the inventive method of preparing the inventivepolymeric carrier cargo complex as defined herein, at least one nucleicacid molecule as defined herein is mixed with the cationic componentsprovided in step b), preferably in the above mentioned ratios.Typically, in the inventive polymeric carrier cargo complex, thecationic components as defined herein, and the at least one nucleic acidmolecule are provided in a molar ratio of about 5 to 10000, preferablyin a molar ratio of about 5 to 5000, more preferably in a molar ratio ofabout 10 to 2500, even more preferably in a molar ratio of about 10 to1000 cationic polymer to nucleic acid. The N/P ratios are preferably asindicated above. In this context it is particularly preferred that theN/P ratios are selected thereby avoiding agglomeration and toxicity invivo.

In a specific embodiment, (AA) components as defined above which do notcomprise —SH moieties can be added in step c) which are therebyincorporated into the the inventive polxmeric carrier cargo complexwithout polymerization by (terminal) —SH moieties. Thereby these (AA)components is/are typically not covalently linked and includednon-covalently in the inventive complex as a further component.

According to a further step d) of the inventive method of preparing theinventive polymeric carrier cargo complex as defined herein, theinventive polymeric carrier cargo complex obtained according to step c)is optionally purified. Purification may occur by using chromatographicmethods, such as HPLC, FPLC, GPS, dialysis, etc.

According to a further step e) of the inventive method of preparing theinventive polymeric carrier cargo complex as defined herein, theinventive polymeric carrier cargo complex obtained according to step c)or d) is optionally lyophilized. For this purpose any suitablecryoprotectant or lyoprotectant may be added to the inventive polymericcarrier cargo complex obtained in step c) or d).

The inventive method of preparing the inventive polymeric carrier cargocomplex as defined herein is particularly suitable to adapt the chemicalproperties of the desired inventive polymeric carrier cargo complex dueto specific selection of its components of the polymeric carrier therebyavoiding agglomeration and toxicity in vivo.

According to a further aspect the present invention also provides amethod for transfecting a cell, a tissue or an organism, therebyapplying or administering the inventive polymeric carrier cargo complexin combination with at least one second nucleic acid molecule,particularly for therapeutic purposes. In this context, typically afterpreparing the inventive polymeric carrier cargo complex as describedabove, the inventive polymeric carrier cargo complex is administered toa cell, a tissue or an organism, in combination with the at least onesecond nucleic acid encoding a protein or peptide as described herein,or as a pharmaceutical composition or vaccine as described herein, morepreferably using any of the administration modes as described herein.The method for transfecting a cell may be carried out in vitro, in vivoor ex vivo.

Likewise, according to another aspect, the present invention alsorelates to the use of the inventive polymeric carrier cargo complexadministered in combination with at least one second nucleic acidmolecule, particularly for therapeutic purposes, for transfecting acell, a tissue or an organism, thereby applying or administering theinventive polymeric carrier cargo complex as described above to a cell,a tissue or an organism in combination with the at least one secondnucleic acid molecule encoding a protein or peptide as described herein,preferably in non-packaged form or as a pharmaceutical composition orvaccine as described herein, more preferably using any of theadministration modes as described herein. The administration may becarried out in vitro, in vivo or ex vivo.

The polymeric carrier cargo complex in combination with the secondnucleic acid molecule, preferably an RNA, as described herein, may alsobe used as a medicament. The polymeric carrier cargo complex incombination with the second nucleic acid molecule, preferably an RNA, asdescribed herein, is preferably used in the treatment or prophylaxis ofa disease selected from a tumour or a cancer disease, an infectiousdisease, an autoimmune disease or an allergy or for use as animmunostimulating agent or adjuvant in the treatment or prophylaxis of adisease selected from a tumour or a cancer disease, a cardiovasculardisease, an infectious disease, an autoimmune disease or an allergy. Thepolymeric carrier cargo complex in combination with the second nucleicacid molecule, preferably an RNA, as described herein, may beadministered, preferably in a safe and effective amount as definedherein, orally, parenterally, by inhalation spray, topically, rectally,nasally, buccally, vaginally or via an implanted reservoir. The termparenteral as used herein includes subcutaneous, intravenous,intramuscular, intraarticular, intranodal, intrasynovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or infusion techniques.Preferably, the polymeric carrier cargo complex and the second nucleicacid molecule encoding a protein or peptide are administeredintramuscularly.

The polymeric carrier cargo complex in combination with the secondnucleic acid molecule, preferably an RNA, as described herein, may beadministered by parenteral injection, more preferably by subcutaneous,intravenous, intramuscular, intraarticular, intranodal, intrasynovial,intrasternal, intrathecal, intrahepatic, intralesional, intracranial,transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or via infusion techniques.Particularly preferred is intradermal and intramuscular injection. Inone particularly preferred embodiment, the polymeric carrier cargocomplex in combination with the second nucleic acid molecule isadministered intramuscularly.

Methods for intramuscular administration are known in the art.Typically, a liquid is injected into a skeletal muscle (such as M.gluteus, M. deltoideus or M. vastus lateralis) using, for example, asyringe or a needle-free injection system, such as a jet injectionsystem.

In a preferred embodiment, the polymeric carrier cargo complexadministered in combination with the second nucleic acid molecule asdefined herein is used as a medicament, immunostimulating agent oradjuvant, wherein the only two active ingredients are the polymericcarrier cargo complex and the second nucleic acid molecule. An “activeingredient”, in this context, may be any compound having a therapeuticeffect, particularly capable of eliciting an immune response or ofstimulating/modulating an immune response.

In a further aspect, the present invention also provides apharmaceutical composition, comprising the inventive polymeric carriercargo complex, which is formulated together with at least one secondnucleic acid molecule encoding a protein or peptide, wherein the secondnucleic acid molecule is preferably an RNA molecule.

In one embodiment, the invention provides a pharmaceutical compositioncomprising:

-   (A) a polymeric carrier cargo complex, comprising:    -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components, and    -   b) as a cargo at least one first nucleic acid molecule,        and-   (B) at least one additional pharmaceutically active component,    preferably a second nucleic acid molecule, wherein the at least one    second nucleic acid molecule is an RNA molecule encoding a protein    or a peptide.

The pharmaceutical composition optionally comprises a pharmaceuticallyacceptable carrier and/or vehicle.

As a first ingredient (component (A)), the inventive pharmaceuticalcomposition comprises the inventive polymeric carrier cargo complexformed by the nucleic acid cargo and the polymeric carrier as definedherein (and, optionally, (AA) component(s)).

As a second ingredient (component (B)), the inventive pharmaceuticalcomposition may comprise at least one additional pharmaceutically activecomponent. A pharmaceutically active component in this connection is acompound that has a therapeutic effect to heal, ameliorate or prevent aparticular indication, preferably cancer diseases, autoimmune disease,allergies or infectious diseases. Such compounds include, withoutimplying any limitation, peptides or proteins, preferably as definedherein, nucleic acids, preferably as defined herein, (therapeuticallyactive) low molecular weight organic or inorganic compounds (molecularweight less than 5000, preferably less than 1000), sugars, antigens orantibodies, preferably as defined herein, therapeutic agents alreadyknown in the prior art, antigenic cells, antigenic cellular fragments,cellular fractions; cell wall components (e.g. polysaccharides),modified, attenuated or de-activated (e.g. chemically or by irradiation)pathogens (virus, bacteria etc.), adjuvants, preferably as definedherein, etc.

In a preferred embodiment, the second ingredient (component (B)) is asecond nucleic acid molecule as defined herein, preferably an RNA, morepreferably an mRNA, encoding a protein or peptide, wherein the proteinor peptide has a therapeutic effect to heal, ameliorate or prevent aparticular indication, preferably cancer diseases, autoimmune disease,allergies or infectious diseases. In this context it is particularlypreferred, that the encoded peptides or proteins are antigenic peptidesor proteins.

In particularly preferred embodiments, the pharmaceutical compositioncomprises as a second ingredient (component (B)) an RNA, preferably anmRNA, preferably as defined herein with respect to the inventivepolymeric carrier cargo complex for use as an immunostimulating agent oras an adjuvant. In particular, any one of the features or anycombination of features described herein with regard to the inventivepolymeric carrier cargo complex for use as an immunostimulating agent oras an adjuvant may likewise be applied to the RNA comprised in theinventive pharmaceutical composition as a second ingredient (component(B)). In particular, the RNA comprised in the inventive pharmaceuticalcomposition as a second ingredient (component (B)) may comprise at leastone selected from the group consisting of a 5′-UTR, a 3′-UTR, a poly(A)sequence, a poly(C) sequence and a histone stem-loop sequence, whereineach of these features is preferably as defined herein.

In a particularly preferred embodiment, the RNA comprised in theinventive pharmaceutical composition as a second ingredient (component(B)) comprises a nucleic acid sequence derived from a 5′-TOP-UTR, aGC-optimized coding sequence, a nucleic acid sequence derived from the3′-UTR of an albumin gene, a poly(A)-sequence, a poly(C)-sequence, and ahistone stem loop, wherein each of these features is preferably asdefined herein.

Preferably, component (B) is not covalently linked, in particular not bya disulfide bond, with component (A). Thus, component (B) is preferablynot covalently linked, such as by a disulfide bond, to the polymericcarrier and/or the at least one nucleic acid molecule of the polymericcarrier cargo complex. Preferably, the at least one second nucleic acidmolecule is not covalently linked to the polymeric carrier cargocomplex, in particular not to the polymeric carrier of the polymericcarrier cargo complex. For example, preferably, the at least one secondnucleic acid molecule, is not covalently linked to the polymeric carriercargo complex, such as to the polymeric carrier, by a disulfide bond.However, in an embodiment, wherein component (A) and component (B) arelinked via disulfide bonds, such linkage is preferably not realized viaa crosslinker, such as via a 3,6-Dioxa-1,8-octanedithiol (DODT)crosslinker. Furthermore, in an embodiment, wherein component (A) andcomponent (B) are linked via disulfide bonds, component (B) ispreferably not ovalbumine or a fragment of ovalbumine. Moreover, in apreferred embodiment, components (A) and (B) do not form a micellestructure together, in particular, the polymeric carrier preferably doesnot form a micelle structure.

Furthermore, the inventive pharmaceutical composition may comprise apharmaceutically acceptable carrier and/or vehicle. In the context ofthe present invention, a pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of the inventive pharmaceuticalcomposition. If the inventive pharmaceutical composition is provided inliquid form, the carrier will typically be pyrogen-free water; isotonicsaline or buffered (aqueous) solutions, e.g phosphate, citrate etc.buffered solutions. The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. liquids occurring in “in vivo” methods, such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer or Ringer-Lactate solution is particularly preferred as a liquidbasis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to apatient to be treated. The term “compatible” as used here means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the inventive polymeric carrier cargocomplex and the at least one additional pharmaceutically activecomponent as defined herein in such a manner that no interaction occurswhich would substantially reduce the pharmaceutical effectiveness of theinventive pharmaceutical composition under typical use conditions.Pharmaceutically acceptable carriers, fillers and diluents must, ofcourse, have sufficiently high purity and sufficiently low toxicity tomake them suitable for administration to a person to be treated. Someexamples of compounds which can be used as pharmaceutically acceptablecarriers, fillers or constituents thereof are sugars, such as, forexample, lactose, glucose and sucrose; starches, such as, for example,corn starch or potato starch; cellulose and its derivatives, such as,for example, sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; tallow; solid glidants,such as, for example, stearic acid, magnesium stearate; calcium sulfate;vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid.

In certain embodiments of the invention, the polymeric carrier cargocomplex comprised in the inventive pharmaceutical composition is used asan adjuvant. For example, it is used as an adjuvant, and/or has adjuvantproperties, as may be readily determined by the person of ordinary skillusing routine methodologies, and including methodologies as describedherein.

As a first ingredient (component (A)) the inventive pharmaceuticalcomposition includes (e.g. as an adjuvant) at least one polymericcarrier cargo complex, comprising

-   -   a) (as a carrier) a polymeric carrier comprising        disulfide-crosslinked cationic components, preferably formed by        disulfide-crosslinked cationic components, and    -   b) (as a cargo) at least one (first) nucleic acid molecule.

According to a specific embodiment, the inventive pharmaceuticalcomposition may comprise an (additional) adjuvant. In this context, anadjuvant may be understood as any compound, which is suitable toinitiate or increase an immune response of the innate immune system,i.e. a non-specific immune response. With other words, whenadministered, the inventive pharmaceutical composition typically elicitsan innate immune response due to the adjuvant, optionally containedtherein. Such an adjuvant may be selected from any adjuvant known to askilled person and suitable for the present case, i.e. supporting theinduction of an innate immune response in a mammal.

In particular, such an adjuvant may be selected from any adjuvant knownto a skilled person and suitable for the present case, i.e. supportingthe induction of an innate immune response in a mammal and/or suitablefor depot and delivery of the components of the inventive pharmaceuticalcomposition. Preferred as adjuvants suitable for depot and delivery arecationic or polycationic compounds as defined above. Likewise, theadjuvant may be selected from the group consisting of, without beinglimited thereto, cationic or polycationic compounds as defined above,from chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution,aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphategel; glucans from algae; algammulin; aluminium hydroxide gel (alum);highly protein-adsorbing aluminium hydroxide gel; low viscosityaluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80(0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4);AVRIDINE™ (propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of:i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo [4,5-c] quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferon gamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT 5 oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalenewater emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™and DMURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™((3-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; micro spheres/nanospheres); PLURONIC L121™; PMMA(polymethylmethacrylate); PODDS™ (proteinoid microspheres); polyethylenecarbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylicacid complex); polysorbate 80 (Tween 80); protein cochleates (AvantiPolar Lipids, Inc., Alabaster, Ala.); STIMULON™ (QS-21); Quil-A (Quil-Asaponin); 5-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c] quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”);Sendai proteoliposomes and Sendai containing lipid matrices; Span-85(sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Aladipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-Lthreonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin, microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, 35 IC31, Imidazoquinolines, Ampligen, Ribi529,IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys,Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides,UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable asantagonists including CGRP neuropeptide.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intraarticular, intranodal, intrasynovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intraarticular, intranodal, intrasynovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or via infusion techniques.Particularly preferred is intradermal and intramuscular injection. Inone particularly preferred embodiment, the pharmaceutical composition isadministered intramuscularly.

Methods for intramuscular administration are known in the art.Typically, a liquid is injected into a skeletal muscle (such as M.gluteus, M. deltoideus or M. vastus lateralis) using, for example, asyringe or a needle-free injection system, such as a jet injectionsystem.

Sterile injectable forms of the inventive pharmaceutical compositionsmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation of the inventivepharmaceutical composition.

The inventive pharmaceutical composition as defined herein may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredients, i.e. the inventivepolymeric carrier cargo complex and the at least one second nucleic acidmolecule, are combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive polymeric carrier cargo complex and the at least one secondnucleic acid molecule suspended or dissolved in one or more carriers.Carriers for topical administration include, but are not limited to,mineral oil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, the inventive pharmaceutical composition can beformulated in a suitable lotion or cream. In the context of the presentinvention, suitable carriers include, but are not limited to, mineraloil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water.

The inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the inventive polymeric carrier cargocomplex and the at least one second nucleic acid molecule as definedherein. As used herein, a “safe and effective amount” means an amount ofthe inventive polymeric carrier cargo complex and the at least onesecond nucleic acid molecule as such that is sufficient to significantlyinduce a positive modification of a disease or disorder as definedherein. At the same time, however, a “safe and effective amount” issmall enough to avoid serious side-effects and to permit a sensiblerelationship between advantage and risk. The determination of theselimits typically lies within the scope of sensible medical judgment. A“safe and effective amount” of the components of the inventivepharmaceutical composition, particularly of the inventive polymericcarrier cargo complex and the at least one second nucleic acid moleculeas defined herein, will furthermore vary in connection with theparticular condition to be treated and also with the age and physicalcondition of the patient to be treated, the body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,the activity of the inventive polymeric carrier cargo complex and the atleast one second nucleic acid molecule, the severity of the condition,the duration of the treatment, the nature of the accompanying therapy,of the particular pharmaceutically acceptable carrier used, and similarfactors, within the knowledge and experience of the accompanying doctor.The inventive pharmaceutical composition may be used for human and alsofor veterinary medical purposes, preferably for human medical purposes,as a pharmaceutical composition in general or as a vaccine,immunostimulating agent or adjuvant.

According to a particular preferred aspect, the inventive pharmaceuticalcomposition (or the inventive polymeric carrier cargo complex) may beprovided or used as an immunostimulating agent. In this context, theinventive pharmaceutical composition is preferably as defined above.More preferably, the nucleic acid of the inventive polymeric carriercargo complex, preferably contained in the pharmaceutical composition,is typically an immunostimulatory nucleic acid as defined herein, e.g. aCpG-DNA or an immunostimulatory RNA (isRNA). Alternatively oradditionally, the nucleic acid of the inventive polymeric carrier cargocomplex, preferably contained in the pharmaceutical composition, is acoding nucleic acid as defined herein, preferably a cDNA or an mRNA,more preferably encoding an adjuvant protein preferably as definedherein.

In a specific embodiment in this context, it is preferred that anadjuvant protein is a component of the inventive polymeric carrier cargocomplex and, preferably, of the polymeric carrier.

According to an even more preferred embodiment, the inventivepharmaceutical composition (or the inventive polymeric carrier cargocomplex) may be provided or used as an adjuvant. In this context, theadjuvant is preferably defined as the inventive pharmaceuticalcomposition above. More preferably, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in the adjuvant,is typically an immunostimulatory nucleic acid as defined herein, e.g. aCpG-DNA or an immunostimulatory RNA (isRNA). Alternatively oradditionally, the nucleic acid of the inventive polymeric carrier cargocomplex, preferably contained in the adjuvant, is a coding nucleic acidas defined herein, preferably a cDNA or an mRNA, more preferablyencoding an adjuvant protein, preferably as defined herein. Theinventive polymeric carrier cargo complex, preferably contained in theadjuvant, typically initiates an innate immune response in the patientto be treated. Such an adjuvant may be utilized in any accompanyingtherapy, with any known vaccine or any further (known) therapeuticagent, preferably prior to, concurrent with or subsequent toadministration of the main therapy, prior to, concurrent with orsubsequent to administration of a further (known) vaccine or a (known)further therapeutic agent.

The polymeric carrier cargo complex, which is administered incombination with a second nucleic acid molecule as described herein, orthe inventive pharmaceutical composition as defined herein provided orused as an adjuvant is preferably capable of triggering anon-antigen-specific, (innate) immune reaction (as provided by theinnate immune system), preferably in an immunostimulating manner. Animmune reaction can generally be brought about in various ways. Animportant factor for a suitable immune response is the stimulation ofdifferent T-cell sub-populations. T-lymphocytes typically differentiateinto two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2(Th2) cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of effector proteins(cytokines) produced by them. Thus, Th1 cells assist the cellular immuneresponse by activation of macrophages and cytotoxic T-cells. Th2 cells,on the other hand, promote the humoral immune response by stimulation ofB-cells for conversion into plasma cells and by formation of antibodies(e.g. against antigens). The Th1/Th2 ratio is therefore of greatimportance in the immune response. In connection with the presentinvention, the Th1/Th2 ratio of the immune response is preferablydisplaced by the immune-stimulating agent, namely the inventivepolymeric carrier cargo complex in the direction towards the cellularresponse, that is to say the Th1 response, and a predominantly cellularimmune response is thereby induced. As defined above, the inventivepolymeric carrier cargo complex exerts by itself an unspecific innateimmune response, which allows the inventive polymeric carrier cargocomplex be used as such (without adding another pharmaceutically activecomponent) as an immunostimulating agent. If administered together withanother pharmaceutically active component, preferably a specificallyimmunogenic component, preferably an antigen and more preferably the atleast one second nucleic acid molecule encoding an antigenic peptide orprotein, the nucleic acid of the polymeric carrier cargo complex servesas an adjuvant supporting the specific adaptive immune response elicitedby the other pharmaceutically active component e.g. an antigen.

In a preferred embodiment, the pharmaceutical composition contains asthe only pharmaceutically active ingredients the polymeric carrier cargocomplex and the second nucleic acid molecule as defined herein. An“active ingredient”, in this context, may be any compound having atherapeutic effect, capable of eliciting an immune response or ofstimulating/modulating an immune response, such as, for instance, anucleic acid encoding a peptide antigen or a protein antigen.

Determination of the Immunostimulatory or Adjuvant Capacity of anInventive Compound or an Inventive Complex:

For the determination of the immunostimulatory capacity of an inventivecompound or an inventive complex several methods are known in the artand may be used. E.g., in vitro methods are advantageous to screen forcompounds as to their capacity to induce cytokines, which are(exclusively or at least typically) part of the innate immune system andthereby (as an additional arm of the immune system) typically improvethe induction of an antigen-specific immune response caused by anantigen. For this purpose, e.g. PBMCs may be isolated from blood samplesand stimulated with the particular compound or complex. Afterincubation, secretion of the desired cytokines (e.g. as a reaction of anactivation of the PAMP receptors) being typically part of the innateimmune system (and not of the antigen-specific immune system) isdetermined by ELISA. These selected cytokines may be used in the art asdeterminants of the induction of an innate immune response in the body.In this context, the secretion of TNF-alpha and IFN-alpha is preferablymeasured to determine the unspecific (innate immune response) evoked bya compound or complex. Especially, IFN-alpha plays an important role inthe induction of an unspecific immune response after viral infection.Accordingly, it is particularly preferred that the the immunostimulatorycompound or complex, which shall be identified by the screening assay,induces the secretion of e.g. IFN-alpha. Such a compound or complex maythen be applied e.g. for the use as an immunostimulating agent invaccination therapies.

IFN-alpha is part of the family of type I interferons. Type Iinterferons (IFN) are pleiotropic cytokines that are essential forsupporting anti-viral immune responses. They induce apoptosis ofvirus-infected cells and cellular resistance to viral infection, inaddition to activating natural killer (NK) and T cells. Type Iinterferons have effects on a large set of cytokines and chemokines thati.a. influence immunocyte maturation, homing, effector functions andapoptosis. Typically, a major role of IFN-α/β is the induction of apriming state affecting the production and regulation of othermediators, including cytokines. For example, IFN-α/β signalingupregulates IFN-γ production by dendritic cells (DCs) and T cells andthereby favours the induction and maintenance of Th1 cells. Shifting ofan immune response in direction of a Th1 immune response may becomeimportant, once protein or peptide vaccines are used, because thesevaccines usually induce a Th2-based immune response which consequentlyprevents the induction of cytotoxic T cells.

Therefore, it is preferred that a compound or complex to be used as anadjuvant may preferably have the property of shifting anantigen-specific immune response caused by a vaccine to a Th1-basedimmune response. The direction of an immune response induced by avaccine is usually measured by determination of the induction of severalsubtypes of antigen-specific antibodies and the induction ofantigen-specific cytotoxic CD8+ T cells. In this context, the subtypeantibody IgG1 represents the induction of a Th2-based immune responseand the induction of the subtype antibody IgG2a and the induction ofcytotoxic T cells represent the induction of a Th1-based immuneresponse. The induction of antigen-specific antibodies is determined bymeasurement of the antibody titer in the blood of the vaccinee by ELISA.The induction of antigen-specific cytotoxic T cells is determined bymeasurement of IFN-gamma secretion in splenocytes after stimulation withantigen-specific peptides by ELISPOT. In this context, the induction ofIFN-gamma secretion proves that antigen-specific cytotoxic T cells arepresent in the spleen which can specifically attack cells which presentepitopes of the antigen on MHC I molecules on their surface.

Thus, for the determination of beneficial properties of an adjuvant invivo vaccinations are performed. Therewith, it is possible to find out,if the adjuvant or immunostimulatory compound or complex improves anantigen-specific immune response caused by the vaccine and, furthermore,if it can shift an antigen-specific immune response in the desireddirection to display adjuvant properties. Particularly, in the inductionof an anti-tumoral immune response the induction of a Th1-shifted immuneresponse, especially the induction of cytotoxic T cells plays a majorrole, because the induction of antigen-specific cytotoxic T cellsrepresents an indispensable prerequisite for the successful combat of atumour.

Accordingly, the methods to screen for compounds or complexes whichactually exhibit properties as immunostimulating agents and/or adjuvantsare well known in the art and may readily be applied e.g. by ELISA testsmeasuring the immune response elicited by the testedcompounds/complexes.

According to another particularly preferred aspect, the inventivepharmaceutical composition (or the inventive polymeric carrier cargocomplex, which is administered in combination with a second nucleic acidmolecule as defined herein) may be provided or used as a vaccine.

In this context, the vaccine is preferably defined as an adjuvant or asan inventive pharmaceutical composition as disclosed above. Morepreferably, the nucleic acid of the inventive polymeric carrier cargocomplex, preferably contained in such a vaccine, may be any nucleic acidas defined above, preferably an immunostimulatory nucleic acid asdefined herein, e.g. a CpG-DNA or an immunostimulatory RNA (isRNA).Alternatively or additionally, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in the vaccine, isa coding nucleic acid as defined herein, preferably a cDNA or an mRNA,more preferably encoding an adjuvant protein, preferably as definedherein. Alternatively or additionally, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in the vaccine, isa coding nucleic acid as defined herein, preferably a cDNA or an mRNA,more preferably encoding an antigen, preferably as defined herein.Furthermore, the vaccine comprises a second nucleic acid molecule,preferably an RNA, encoding a protein or peptide as defined herein. Inaddition, the inventive vaccine may contain an antigen, preferably asdefined above, as a protein or peptide, or antigenic cells, antigeniccellular fragments, cellular fractions; cell wall components (e.g.polysaccharides), modified, attenuated or de-activated (e.g. chemicallyor by irradiation) pathogens (virus, bacteria etc.).

As described above, the present invention provides a polymeric carriercargo complex for use as an immunostimulating agent or an adjuvant,wherein the polymeric carrier cargo complex is administered incombination with at least one second nucleic acid molecule encoding aprotein or a peptide, wherein the polymeric carrier cargo complex andthe second nucleic acid molecule are administered intramuscularly andwherein the polymeric carrier cargo complex and the second nucleic acidmolecule are preferably not administered together with a protein orpeptide antigen selected from the group consisting of an antigen from apathogen associated with infectious disease, an antigen associated withallergy or allergic disease, an antigen associated with autoimmunedisease, an antigen associated with a cancer or tumour disease, or afragment, variant and/or derivative of said protein or peptide antigen.According to a preferred embodiment, the inventive pharmaceuticalcomposition or the inventive vaccine do likewise not comprise a proteinor peptide antigen selected from the group consisting of an antigen froma pathogen associated with infectious disease, an antigen associatedwith allergy or allergic disease, an antigen associated with autoimmunedisease, an antigen associated with a cancer or tumour disease, or afragment, variant and/or derivative of said protein or peptide antigen.More preferably, the inventive pharmaceutical composition or theinventive vaccine does not comprise a protein or peptide antigen.

According to a first embodiment such an inventive vaccine supports orelicits an innate immune response of the immune system of a patient tobe treated, preferably due to an immunostimulatory capacity of theinventive polymeric carrier cargo complex.

According to a second embodiment, the inventive vaccine may furtherelicit an adaptive immune response, preferably due to the protein orpeptide encoded by the second nucleic acid molecule as defined herein,which is suitable to elicit an adaptive immune response. Alternatively,an additional antigen can be provided in form of a peptide, a protein oran epitope of said antigen. The antigen may also be a component of theinventive polymeric carrier, e.g. as a (AA) component, as definedherein.

The inventive vaccine, pharmaceutical composition, immunostimulatingagent or adjuvant may also comprise a pharmaceutically acceptablecarrier, adjuvant, and/or vehicle as defined herein for the inventivepharmaceutical composition. In the specific context of the inventivevaccine, the choice of a pharmaceutically acceptable carrier isdetermined in principle by the manner in which the inventive vaccine isadministered. The inventive vaccine can be administered, for example,systemically or locally. Routes for systemic administration in generalinclude, for example, transdermal, oral, parenteral routes, includingsubcutaneous, intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.

Intramuscular administration, e.g. via needle injection or needle-freeinjection (e.g. jet injection), is particularly preferred.

Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route, most preferably by intramuscular route. Inventivevaccines are therefore preferably formulated in liquid (or sometimes insolid) form. The suitable amount of the inventive vaccine to beadministered can be determined by routine experiments with animalmodels. Such models include, without implying any limitation, rabbit,sheep, mouse, rat, dog and non-human primate models. Preferred unit doseforms for injection include sterile solutions of water, physiologicalsaline or mixtures thereof. The pH of such solutions should be adjustedto about 7.4. Suitable carriers for injection include hydrogels, devicesfor controlled or delayed release, polylactic acid and collagenmatrices. Suitable pharmaceutically acceptable carriers for topicalapplication include those, which are suitable for use in lotions,creams, gels and the like. If the inventive vaccine is to beadministered orally, tablets, capsules and the like are the preferredunit dose form. The pharmaceutically acceptable carriers for thepreparation of unit dose forms which can be used for oral administrationare well known in the prior art. The choice thereof will depend onsecondary considerations such as taste, costs and storability, which arenot critical for the purposes of the present invention, and can be madewithout difficulty by a person skilled in the art.

The inventive vaccine, pharmaceutical composition, immunostimulatingagent or adjuvant can additionally contain one or more auxiliarysubstances in order to increase its immunogenicity or immunostimulatorycapacity, if desired. A synergistic action of the inventive polymericcarrier cargo complex and the second nucleic acid molecule as definedherein and of an auxiliary substance, which may be optionally containedin the inventive vaccine, pharmaceutical composition, immunostimulatingagent or adjuvant as defined herein, is preferably achieved thereby.Depending on the various types of auxiliary substances, variousmechanisms can come into consideration in this respect. For example,compounds that permit the maturation of dendritic cells (DCs), forexample lipopolysaccharides, TNF-alpha or CD40 ligand, form a firstclass of suitable auxiliary substances. In general, it is possible touse as auxiliary substance any agent that influences the immune systemin the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, suchas GM-CFS, which allow an immune response to be enhanced and/orinfluenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that further promote the innate immune response, such asIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32,IL-33, INF-alpha, IFN-beta, INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta orTNF-alpha, growth factors, such as hGH.

Further additives which may be included in the inventive vaccine,pharmaceutical composition, immunostimulating agent or adjuvant areemulsifiers, such as, for example, Tween®; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

The inventive vaccine, pharmaceutical composition, immunostimulatingagent or adjuvant can also additionally contain any further compound,which is known to be immunostimulating due to its binding affinity (asligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (asligands) to murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

The inventive vaccine, pharmaceutical composition, immunostimulatingagent or adjuvant can also additionally or alternatively contain animmunostimulatory RNA, i.e. an RNA derived from an immunostimulatoryRNA, which triggers or increases an (innate) immune response.Preferably, such an immunostimulatory RNA may be in general be asdefined hereinbefore.

Another class of compounds, which may be added to an inventive vaccine,pharmaceutical composition, immunostimulating agent or adjuvant in thiscontext, may be CpG nucleic acids, in particular CpG-RNA or CpG-DNA. ACpG-RNA or CpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), adouble-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA)or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid ispreferably in the form of CpG-RNA, more preferably in the form ofsingle-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid preferablycontains at least one or more (mitogenic) cytosine/guanine dinucleotidesequence(s) (CpG motif(s)). According to a first preferred alternative,at least one CpG motif contained in these sequences, that is to say theC (cytosine) and the G (guanine) of the CpG motif, is unmethylated. Allfurther cytosines or guanines optionally contained in these sequencescan be either methylated or unmethylated. According to a furtherpreferred alternative, however, the C (cytosine) and the G (guanine) ofthe CpG motif can also be present in methylated form.

The present invention furthermore provides several applications and usesof the inventive polymeric carrier cargo complex, which is administeredin combination with a second nucleic acid molecule encoding a protein orpeptide, as defined herein, the inventive pharmaceutical composition,the inventive immunostimulating agent or adjuvant and the inventivevaccine comprising same or of kits comprising same.

According to one specific embodiment, the present invention is directedto the first medical use of the inventive polymeric carrier cargocomplex in combination with a second nucleic acid molecule as definedherein as a medicament, preferably as an immunostimulating agent,adjuvant or vaccine.

According to another embodiment, the present invention is directed tothe second medical use of the inventive polymeric carrier cargo complexadministered in combination with at least one second nucleic acidmolecule as defined herein, for the treatment of diseases as definedherein, preferably to the use of the inventive polymeric carrier cargocomplex in combination with a second nucleic acid molecule as definedherein, of a pharmaceutical composition, vaccine, immunostimulatingagent, adjuvant or vaccine comprising same or of kits comprising samefor the preparation of a medicament for the prophylaxis, treatmentand/or amelioration of various diseases as defined herein, particularlyprophylaxis, treatment and/or amelioration of such diseases as definedherein. Preferably, the pharmaceutical composition, an immunostimulatingagent, an adjuvant or a vaccine is used or administered to a patient inneed thereof for this purpose.

Preferably, diseases as mentioned herein are selected from cancer ortumour diseases, infectious diseases, preferably (viral, bacterial orprotozoan) infectious diseases, autoimmune diseases, allergies orallergic diseases, monogenetic diseases, i.e. (hereditary) diseases, orgenetic diseases in general, diseases which have a genetic inheritedbackground and which are typically caused by a single gene defect andare inherited according to Mendel's laws, cardiovascular diseases,neuronal diseases or any disease which can be influenced by the presentinvention.

Such diseases include cancer or tumor diseases, preferably selected frommelanomas, malignant melanomas, colon carcinomas, lymphomas, sarcomas,blastomas, renal carcinomas, gastrointestinal tumors, gliomas, prostatetumors, bladder cancer, rectal tumors, stomach cancer, oesophagealcancer, pancreatic cancer, liver cancer, mammary carcinomas (=breastcancer), uterine cancer, cervical cancer, acute myeloid leukaemia (AML),acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chroniclymphocytic leukaemia (CLL), hepatomas, various virus-induced tumorssuch as, for example, papilloma virus-induced carcinomas (e.g. cervicalcarcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumors(e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma), heptatitisB-induced tumors (hepatocell carcinomas), HTLV-1- and HTLV-2-inducedlymphomas, acoustic neuroma, lung carcinomas (=lung cancer=bronchialcarcinoma), small-cell lung carcinomas, pharyngeal cancer, analcarcinoma, glioblastoma, rectal carcinoma, astrocytoma, brain tumors,retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginalcancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome,meningiomas, Schneeberger disease, hypophysis tumor, Mycosis fungoides,carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer,renal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin'slymphomas, urethral cancer, CUP syndrome, head/neck tumors,oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma,oesophageal carcinoma (=oesophageal cancer), wart involvement, tumors ofthe small intestine, craniopharyngeomas, ovarian carcinoma, genitaltumors, ovarian cancer (=ovarian carcinoma), pancreatic carcinoma(=pancreatic cancer), endometrial carcinoma, liver metastases, penilecancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytoma, lidtumor, prostate cancer (=prostate tumors), etc.

According to one further specific embodiment, diseases as defined hereincomprise infectious diseases, preferably (viral, bacterial orprotozoological) infectious diseases. Such infectious diseases, arepreferably caused by a viral, bacterial, fungal or protozoan pathogen,preferably selected from the pathogens Acinetobacter baumannii,Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense,Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascarislumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillusanthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystishominis, Blastomyces dermatitidis, Bordetella pertussis, Borreliaburgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugiamalayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderiaspecies, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridaefamily, Campylobacter genus, Candida albicans, Candida spp, Chlamydiatrachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJDprion, Clonorchis sinensis, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, Clostridium perfringens, Clostridiumspp, Clostridium tetani, Coccidioides spp, coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus,Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4),Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichiachaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica,Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie Avirus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus(EBV), Escherichia coli O157:H7, O111 and O104:H4, Fasciola hepatica andFasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,Francisella tularensis, Fusobacterium genus, Geotrichum candidum,Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis BVirus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, Herpessimplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV(Human immunodeficiency virus), Hortaea werneckii, Human bocavirus(HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7),Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus,Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassavirus, Legionella pneumophila, Leishmania genus, Leptospira genus,Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV),Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Naegleria fowleri, Necator americanus, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp,Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family,Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani,Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystisjirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV),Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsiaprowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fevervirus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus,Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus,Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcusgenus, Staphylococcus genus, Streptococcus agalactiae, Streptococcuspneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taeniagenus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocaracanis or Toxocara cati, Toxoplasma gondii, Treponema pallidum,Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuristrichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasmaurealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV),Variola major or Variola minor, vCJD prion, Venezuelan equineencephalitis virus, Vibrio cholerae, West Nile virus, Western equineencephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersiniaenterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis. Inthis context, an infectious disease, preferably a viral, bacterial orprotozoan infectious diseases, is typically selected from influenza,malaria, SARS, yellow fever, AIDS, Lyme borreliosis, Leishmaniasis,anthrax, meningitis, viral infectious diseases such as AIDS, Condylomaacuminata, hollow warts, Dengue fever, three-day fever, Ebola virus,cold, early summer meningoencephalitis (FSME), flu, shingles, hepatitis,herpes simplex type I, herpes simplex type II, Herpes zoster, influenza,Japanese encephalitis, Lassa fever, Marburg virus, measles,foot-and-mouth disease, mononucleosis, mumps, Norwalk virus infection,Pfeiffer's glandular fever, smallpox, polio (childhood lameness),pseudo-croup, fifth disease, rabies, warts, West Nile fever, chickenpox,cytomegalic virus (CMV), bacterial infectious diseases such asmiscarriage (prostate inflammation), anthrax, appendicitis, borreliosis,botulism, Camphylobacter, Chlamydia trachomatis (inflammation of theurethra, conjunctivitis), cholera, diphtheria, donavanosis,epiglottitis, typhus fever, gas gangrene, gonorrhoea, rabbit fever,Heliobacter pylori, whooping cough, climatic bubo, osteomyelitis,Legionnaire's disease, leprosy, listeriosis, pneumonia, meningitis,bacterial meningitis, anthrax, otitis media, Mycoplasma hominis,neonatal sepsis (Chorioamnionitis), noma, paratyphus, plague, Reiter'ssyndrome, Rocky Mountain spotted fever, Salmonella paratyphus,Salmonella typhus, scarlet fever, syphilis, tetanus, tripper,tsutsugamushi disease, tuberculosis, typhus, vaginitis (colpitis), softchancre, and infectious diseases caused by parasites, protozoa or fungi,such as amoebiasis, bilharziosis, Chagas disease, Echinococcus, fishtapeworm, fish poisoning (Ciguatera), fox tapeworm, athlete's foot,canine tapeworm, candidosis, yeast fungus spots, scabies, cutaneousLeishmaniosis, lambliasis (giardiasis), lice, malaria, microscopy,onchocercosis (river blindness), fungal diseases, bovine tapeworm,schistosomiasis, porcine tapeworm, toxoplasmosis, trichomoniasis,trypanosomiasis (sleeping sickness), visceral Leishmaniosis,nappy/diaper dermatitis or miniature tapeworm.

According to another specific embodiment, diseases as defined hereincomprise autoimmune diseases as defined in the following. Autoimmunediseases can be broadly divided into systemic and organ-specific orlocalised autoimmune disorders, depending on the principalclinico-pathologic features of each disease. Autoimmune diseases may bedivided into the categories of systemic syndromes, including systemiclupus erythematosus (SLE), Sjögren's syndrome, Scleroderma, RheumatoidArthritis and polymyositis or local syndromes which may beendocrinologic (type I diabetes (Diabetes mellitus Type 1), Hashimoto'sthyroiditis, Addison's disease etc.), dermatologic (pemphigus vulgaris),haematologic (autoimmune haemolytic anaemia), neural (multiplesclerosis) or can involve virtually any circumscribed mass of bodytissue. The autoimmune diseases to be treated may be selected from thegroup consisting of type I autoimmune diseases or type II autoimmunediseases or type III autoimmune diseases or type IV autoimmune diseases,such as, for example, multiple sclerosis (MS), rheumatoid arthritis,diabetes, type I diabetes (Diabetes mellitus Type 1), chronicpolyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis,colitis ulcerosa, type I allergy diseases, type II allergy diseases,type III allergy diseases, type IV allergy diseases, fibromyalgia, hairloss, Bechterew's disease, Crohn's disease, Myasthenia gravis,neurodermitis, Polymyalgia rheumatica, progressive systemic sclerosis(PSS), Reiter's syndrome, rheumatic arthritis, psoriasis, vasculitis,etc, or type II diabetes. While the exact mode as to why the immunesystem induces an immune reaction against autoantigens has not beenelucidated so far, there are several findings with regard to theetiology. Accordingly, the autoreaction may be due to a T-Cell bypass. Anormal immune system requires the activation of B-cells by T-cellsbefore the former can produce antibodies in large quantities. Thisrequirement of a T-cell can be by-passed in rare instances, such asinfection by organisms producing super-antigens, which are capable ofinitiating polyclonal activation of B-cells, or even of T-cells, bydirectly binding to the ß-subunit of T-cell receptors in a non-specificfashion. Another explanation deduces autoimmune diseases from aMolecular Mimicry. An exogenous antigen may share structuralsimilarities with certain host antigens; thus, any antibody producedagainst this antigen (which mimics the self-antigens) can also, intheory, bind to the host antigens and amplify the immune response. Themost striking form of molecular mimicry is observed in Group Abeta-haemolytic streptococci, which shares antigens with humanmyocardium, and is responsible for the cardiac manifestations ofrheumatic fever.

Additionally, according to one further specific embodiment, diseases asdefined herein comprise allergies or allergic diseases, i.e. diseasesrelated to allergies. Allergy is a condition that typically involves anabnormal, acquired immunological hypersensitivity to certain foreignantigens or allergens, such as the allergy antigens as defined herein.Such allergy antigens or allergens may be selected from allergy antigensas defined herein antigens derived from different sources, e.g. fromanimals, plants, fungi, bacteria, etc. Allergens in this context includee.g. grasses, pollens, molds, drugs, or numerous environmental triggers,etc. Allergies normally result in a local or systemic inflammatoryresponse to these antigens or allergens and lead to immunity in the bodyagainst these allergens. Without being bound to theory, severaldifferent disease mechanisms are supposed to be involved in thedevelopment of allergies. According to a classification scheme by P.Gell and R. Coombs the word “allergy” was restricted to type Ihypersensitivities, which are caused by the classical IgE mechanism.Type I hypersensitivity is characterised by excessive activation of mastcells and basophils by IgE, resulting in a systemic inflammatoryresponse that can result in symptoms as benign as a runny nose, tolife-threatening anaphylactic shock and death. Well known types ofallergies include, without being limited thereto, allergic asthma(leading to swelling of the nasal mucosa), allergic conjunctivitis(leading to redness and itching of the conjunctiva), allergic rhinitis(“hay fever”), anaphylaxis, angiodema, atopic dermatitis (eczema),urticaria (hives), eosinophilia, respiratory, allergies to insectstings, skin allergies (leading to and including various rashes, such aseczema, hives (urticaria) and (contact) dermatitis), food allergies,allergies to medicine, etc. Treatment of such allergic disorders ordiseases may occur preferably by desensitizing the immune reaction whichtriggers a specific immune response. Such a desensitizing may be carriedout by administering an effective amount of the allergen or allergicantigen encoded by the nucleic acid as defined herein, preferably, whenformulated as a pharmaceutical composition, to induce a slight immunereaction. The amount of the allergen or allergic antigen may then beraised step by step in subsequent administrations until the immunesystem of the patient to be treated tolerates a specific amount ofallergen or allergic antigen.

In a further aspect, the inventive polymeric carrier cargo complex whichis administered in combination with a second nucleic acid molecule maybe used for the preparation of a pharmaceutical composition, animmunostimulating agent, an adjuvant or a vaccine.

The inventive pharmaceutical composition, immunostimulating agent,adjuvant or vaccine may furthermore be used for the prophylaxis ortreatment of a disease or a disorder as defined herein.

According to a final aspect, the present invention also provides kits,particularly kits of parts, comprising as components alone or incombination with further ingredients at least one inventive polymericcarrier cargo complex which is administered in combination with a secondnucleic acid molecule as defined herein, at least one pharmaceuticalcomposition, immunostimulating agent, adjuvant or vaccine comprisingsame and/or kits comprising same, and optionally technical instructionswith information on the administration and dosage of the polymericcarrier molecule, the nucleic acid, the inventive polymeric carriercomplex, and/or the inventive pharmaceutical composition. Such kits,preferably kits of parts, may be applied, e.g., for any of the abovementioned applications or uses. Such kits, when occurring as a kit ofparts, may further contain each component of the inventivepharmaceutical composition, immunostimulating agent, adjuvant or vaccinein a different part of the kit. Preferably, at least one component ispresent in lyophilized form.

The inventive kit may thus comprise the pharmaceutical compositionand/or the vaccine as described herein, and optionally a liquid vehiclefor solubilising and optionally technical instructions with informationon the administration and dosage of the active composition and/or thevaccine. In a preferred embodiment, the kit comprises a Ringer-lactatesolution.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other, wheresuitable. Furthermore, the term “comprising” shall not be construed asmeaning “consisting of”, if not specifically mentioned. However, in thecontext of the present invention, term “comprising” may be substitutedwith the term “consisting of”, where suitable.

FIGURES

The figures shown in the following are merely illustrative and shalldescribe the present invention in a further way. These figures shall notbe construed to limit the present invention thereto.

FIG. 1: G/C-enriched mRNA sequence R2564 coding for the hemagglutinin(HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)),corresponding to SEQ ID NO: 384.

FIG. 2: RNA sequence of the non-coding immunostimulatory RNA R2025,corresponding to SEQ ID NO: 385.

FIG. 3: FIG. 3 shows that intramuscular vaccination with a combinationof HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargocomplex (R2391, prepared according to Example 1) induces higher titersof antibodies against HA protein compared to vaccination with HA-mRNA(R2564) alone.

-   -   Balb/c mice (n=8 per group) were vaccinated intramuscularly on        days 0 and 14 either with 40 μg HA-mRNA (R2564, SEQ ID NO: 384,        “naked HA-RNA”) alone or with 40 μg HA-mRNA co-formulated with        40 μg of the polymeric carrier cargo complex (R2391,        “RNAdjuvant”). Buffer treated mice served as negative controls.        Induction of functional humoral responses was analysed on day 28        by determining the hemagglutination inhibition (HI) antibody        titer, which is generally used as a surrogate marker of immune        protection against influenza virus infection. A HI titer of 1:40        or greater is typically considered to confer protection. The        experiment was performed as described in Example 2.    -   As can be seen in FIG. 3, all mice vaccinated with the        co-formulation developed HI-titers ≥1:40. In contrast, only 50%        of mice vaccinated with HA-mRNA alone showed HI-titers ≥1:40.    -   Each dot represents an individual animal and the horizontal        lines represent median values.

FIG. 4: FIG. 4 shows that intramuscular vaccination with a combinationof HA-mRNA

(R2564, SEQ ID NO: 384) and the polymeric carrier cargo complex (R2391,prepared according to Example 1) leads to a significant increase in thenumber of central memory CD8+ cells.

-   -   Balb/c mice (n=8 per group) were vaccinated intramuscularly on        days 0 and 14 with either 40 μg HA mRNA (R2564, SEQ ID NO: 384,        “naked HA-RNA”) alone or with 40 μg HA-mRNA co-formulated with        40 μg of the polymeric carrier cargo complex (R2391,        “RNAdjuvant”). Buffer treated mice served as negative controls.        Induction of memory T cell responses in the bone marrow was        analysed 7 weeks after boost vaccination. The experiment was        performed as described in Example 2.    -   As can be seen in FIG. 4, vaccination with the co-formulation        led to a significant increase in the number of central memory        CD8+ T cells compared to mice vaccinated with HA-mRNA alone.    -   Each dot represents an individual animal and the horizontal        lines represent median values. Statistical assessment was        performed with the unpaired t-test (**: p=0.0022; ****:        p<0.0001).

FIG. 5: FIG. 5 shows that intramuscular vaccination with a combinationof HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargocomplex (R2391, prepared according to Example 1) leads to significantincrease in the number of central memory CD4+ cells.

-   -   Balb/c mice (n=8 per group) were vaccinated intramuscularly on        days 0 and 14 with either 40 μg HA mRNA (R2564, SEQ ID NO: 384,        “naked HA-RNA”) alone or with 40 μg HA-mRNA co-formulated with        40 μg of the polymeric carrier cargo complex (R2391,        “RNAdjuvant”). Buffer treated mice served as negative controls.        Induction of memory T cell responses in the bone marrow was        analysed 7 weeks after boost vaccination. The experiment was        performed as described in Example 2.    -   As can be seen in FIG. 5, vaccination with the co-formulation        led to a significant increase in the number of central memory        CD4+ T cells compared to mice vaccinated with HA-mRNA alone.    -   Each dot represents an individual animal and the horizontal        lines represent median values. Statistical assessment was        performed with the unpaired t-test (**: p=0.0010; ****:        p<0.0001).

FIG. 6: FIG. 6 shows that the intramuscular vaccination with acombination of HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carriercargo complex (R2391, prepared according to Example 1) leads tosignificant increase in the number of multifunctional CD4+ T cells.

-   -   Balb/c mice (n=8 per group) were vaccinated intramuscularly on        days 0 and 14 with either 40 μg HA-mRNA (R2564, SEQ ID NO: 384,        “naked HA-RNA”) alone or 40 μg HA-mRNA co-formulated with 40 μg        of the polymeric carrier cargo complex (R2391, “RNAdjuvant”).        Buffer treated mice served as negative controls. Induction of        IFNγ/TNFα double-positive multifunctional CD4+ T cells in the        spleen was analysed 7 weeks after boost vaccination by        intracellular cytokine staining as described in Example 2.    -   As can be seen in FIG. 6, vaccination with the co-formulation        led to a significant increase in the number of multifunctional        CD4+ T cells compared to mice vaccinated with HA-mRNA alone.    -   Each dot represents an individual animal and the horizontal        lines represent median values. Statistical assessment was        performed with the unpaired t-test (***: p=0.0003; **: p<0.007).

FIG. 7: FIG. 7 shows that the intramuscular vaccination with acombination of the HA-mRNA (R2564, SEQ ID NO: 384) and the polymericcarrier cargo complex (R2391, prepared according to Example 1) leads tosignificant increase in the number of effector CD4+ T cells.

-   -   Balb/c mice (n=8 per group) were vaccinated intramuscularly on        days 0 and 14 either with 40 μg HA-mRNA alone (R2564, SEQ ID NO:        384, “naked HA-RNA”) or with 40 μg HA-mRNA co-formulated with 40        μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”).        Buffer treated mice served as negative controls. Induction of        IFNγ/TNFα double-positive multifunctional CD4+ T cells in the        spleen was analysed 7 days after boost vaccination by        intracellular cytokine staining as described in Example 3.    -   As can be seen in FIG. 7, vaccination with the co-formulation        led to a significant increase in the number of multifunctional        CD4+ T cells compared to mice vaccinated with HA-mRNA alone.    -   Each dot represents an individual animal and the horizontal        lines represent median values. Statistical assessment was        performed with the unpaired t-test (*: p=0.0286; **: p=0.0022).

FIG. 8: shows that intramuscular vaccination of domestic pigs with acombination of HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carriercargo complex (R2391, RNAdjuvant; prepared according to Example 1)induces higher titers of antibodies against HA protein compared tovaccination with HA-mRNA vaccine (R2630 RNActive®) alone. This effect isalso detectable with enzymatically polyadenylated mRNA (R2564pA).

-   -   Domestic pigs (n=5 per group) were vaccinated intramuscularly on        days 1 and 29 either with 200 μg HA-mRNA vaccine (R2630        RNActive®) or R2564pA (SEQ ID NO: 384, “naked polyadenylated        HA-RNA”) alone or a co-formulation of R2564 or R2564pA and 200        μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”).        Pre-immune sera served as negative controls. Induction of        functional humoral responses was analysed on day 57 by        determining the hemagglutination inhibition (HI) antibody titer,        which is generally used as a surrogate marker of immune        protection against influenza virus infection. A HI titer of 1:40        or greater is typically considered to confer protection. The        experiment was performed as described in Example 4.    -   As can be seen in FIG. 8, the co-formulation with RNAdjuvant        increased the functional antibodies compared to an mRNA vaccine        (RNActive®) or compared to naked polyadenylated mRNA.    -   Each dot represents an individual animal, the horizontal lines        represent median values.

FIG. 9: shows that intramuscular vaccination of mice with a combinationof RAV-G mRNA (R2506, SEQ ID NO: 391, and R3344) and the polymericcarrier cargo complex (R2391, RNAdjuvant; prepared according toExample 1) induces higher virus neutralization titers compared tovaccination with RAV-G-mRNA alone.

-   -   Balb/c mice (n=8 per group) were vaccinated intramuscularly on        days 0 and 21 either with 20 μg RAV-G mRNA alone (R2506, SEQ ID        NO: 391, “naked RAV-G RNA”; or R3344; enzymatically        polyadenylated naked RAV-G mRNA) or with 20 μg RAV-G mRNA        co-formulated with 40 μg of the polymeric carrier cargo complex        (R2391, “RNAdjuvant”). Buffer treated mice served as negative        controls. Induction of virus neutralization titers was analysed        7 days after boost vaccination as described in Example 5.        According to WHO guidelines, virus neutralization titers of ≤0.5        IU/ml are regarded as protective titers.    -   As can be seen in FIG. 9, vaccination with the co-formulation        led to increased functional antibody titers.    -   Each dot represents an individual animal, the horizontal lines        represent median values.

FIG. 10: shows that intramuscular vaccination of cotton rats with RSV-FmRNA (R2682; HRSV(Long-VR26)-Fdel554-574 mutant, SEQ ID NO: 392) incombination with the polymeric carrier cargo complex (R2391,“RNAdjuvant”) significantly reduce lung titers in cotton rats challengedwith RSV virus compared to vaccination with RSV-F mRNA alone.

-   -   The experiment was performed as described in Example 6.

FIG. 11: G/C-enriched mRNA sequence R2506 (SEQ ID NO: 391) encoding theRAV-G protein.

FIG. 12: G/C-enriched mRNA sequence R2682 (SEQ ID NO: 392) encoding theRSV-F protein (HRSV(Long-VR26)Fdel554-574).

EXAMPLES

The following examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

Example 1: Preparation of the RNA

1. Preparation of DNA and mRNA Constructs

For the present example, a DNA sequence encoding the hemagglutinin (HA)protein of influenza A virus (A/Netherlands/602/2009(H1N1)) was preparedand used for subsequent in vitro transcription reactions.

According to a first preparation, the DNA sequence coding for the abovementioned mRNA was prepared. The construct R2564 (SEQ ID NO: 384) wasprepared by introducing a 5′-TOP-UTR derived from the ribosomal protein32L, modifying the wild type coding sequence by introducing aGC-optimized sequence for stabilization, followed by a stabilizingsequence derived from the albumin-3′-UTR, a stretch of 64 adenosines(poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and ahistone stem loop. In SEQ ID NO: 384 (see FIG. 1) the sequence of thecorresponding mRNA is shown.

For further examples, DNA sequences encoding the RAV-G protein of RabiesVirus and the RSV F protein (HRSV(Long-VR26)Fdel554-574) were preparedas already exemplified for mRNA coding for the hemagglutinin (HA)protein of influenza A virus (A/Netherlands/602/2009(H1N1)) and used forsubsequent in vitro transcription reactions. The corresponding mRNAsequences (SEQ ID NOs 391 and 392) are shown in FIGS. 11 and 12.

2. Preparation of DNA and Non-Coding Immunostimulatory RNA Constructs

For the present example a DNA sequence encoding the non-codingimmunostimulatory RNA (isRNA) R2025 was prepared and used for subsequentin vitro transcription reactions.

According to a first preparation, the DNA sequence coding for the abovementioned RNA was prepared. In SEQ ID NO: 385 (see FIG. 2) the sequenceof the corresponding RNA is shown.

TABLE 1 RNA constructs RNA Description FIG. SEQ ID NO. R2564 InfluenzaHA encoding mRNA 1 SEQ ID NO. 384 R2630 R2025 Non-codingimmunostimulatory 2 SEQ ID NO. 385 R2391 RNA R2506 RAV-G encoding mRNA11 SEQ ID NO. 391 R2682 RSV-F encoding mRNA SEQ ID NO. 392(HRSV(Long-VR26)Fdel554-574)

3. In Vitro Transcription

The respective DNA plasmids prepared according to section 1 above weretranscribed in vitro using T7 polymerase. The in vitro transcription ofinfluenza HA encoding R2564, RAV-G encoding R2506 and R3344 or RSV Fencoding R2682, respectively, was performed in the presence of a CAPanalog (m⁷GpppG). The isRNA R2025 was prepared without CAP analog.Subsequently the RNA was purified using PureMessenger® (CureVac,Tubingen, Germany; WO2008/077592A1).

Enzymatic Adenylation

RNA was reacted with E. coli poly(A) polymerase (Cellscript) using 1 mMATP at 37° C. for 30 or 60 min. Immediately afterwards, RNA was purifiedby precipitation with lithium chloride (incubation for 1 h at −20° C.).The pellet was then washed with cold 75% ethanol and was finallyresolved in water. RNA was run on a gel to assess RNA extension.

4. Preparation of the (Adjuvant) Polymeric Cargo Complex Complex(RNAdjuvant®)

Cationic peptide as cationic component of the polymeric carrier:

CR₁₂C: (SEQ ID NO: 6) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (Cys-Arg₁₂-Cys)

Synthesis of Polymeric Carrier Cargo Complexes:

An RNA molecule having the RNA sequence R2025 as defined in section 2above was mixed with the cationic CR₁₂C peptide component as definedabove. The specified amount of the RNA was mixed with the respectivecationic component in mass ratios as indicated, thereby forming acomplex. If polymerizing cationic components were used according to thepresent invention, polymerization of the cationic components took placesimultaneously to complexation of the nucleic acid cargo. Afterwards,the resulting solution was adjusted with water to a final volume of 50μl and incubated for 30 minutes at room temperature. Further details aredescribed in WO2012013326.

The mass ratio of peptide:RNA was 1:3,7. The polymeric carrier cargocomplex is formed by the disulfide-crosslinked cationic peptide CR12C ascarrier and the immunostimulatory R2025 as nucleic acid cargo. Thispolymeric carrier cargo complex R2025/CR12C (designated R2391) was usedas adjuvant in the following examples. It is also referred to as‘RNAdjuvant®’.

5. Preparation of the Vaccine

The naked mRNAs R2564, R2506, R3344, and R2682 were administered inRinger's Lactate solution. The lyophilyzed polymeric carrier cargocomplex R2391 was dissolved in Ringer's Lactate solution to a finalconcentration of 1 □μg/μl. The co-formulation of naked mRNA R2564,R2506, R3344, or R2682 and R2391 was generated by mixing both componentsdirectly before administration.

For protamine-complexation, the mRNA R2564 was complexed with protaminein a mass ratio of 2:1. After incubation the same amount of naked mRNAR2564 was added to the nanoparticles. This vaccine formulation isreferred to as R2630 RNActive®.

Example 2: Induction of a Humoral and Cellular Immune Response AgainstHemagglutinin of Influenza Virus after Intramuscular Vaccination of MiceImmunization:

On day zero, BALB/c mice were intramuscularly (i.m.) injected into bothM. tibialis with the influenza HA-encoding mRNA (R2564) alone or incombination the polymeric carrier cargo complex (R2391) as shown inTable 2. Therein, the indicated amount in μg refers to the mass of thenucleic acid molecule per se, i.e. in the case of group 3, for instance,where the polymeric carrier cargo complex R2391 is used, animalsreceived a polymeric carrier cargo complex (R2391), which comprised 20μg of RNA. Mice injected with Ringer Lactate (RiLa) buffer served ascontrols. All animals received boost injections on day 14. Blood sampleswere collected on day 28 for the determination serum anti-HA antibodytiters in the hemagglutination inhibition assay. Spleens and bone marrowwere collected on day 45.

TABLE 2 Animal groups Polymeric carrier HA cargo Strain No. Route RiLaRNA complex Vaccination Group sex mice volume buffer R2564 R2391schedule 1 BALB/c 8 i.m. 2 × 30 — — d0: prime, Female 2 × 30 μl d14:boost μl 2 BALB/c 8 i.m. — 2 × 20 — d0: prime, Female 2 × 30 μg d14:boost μl 3 BALB/c 8 i.m. — 2 × 20 2 × 20 μg d0: prime, Female 2 × 30 μgd14: boost μl

Protocols Hemagglutination Inhibition Assay

For hemagglutination inhibition (HI) assay mouse sera were heatinactivated (56° C., 30 min), incubated with kaolin, and pre-adsorbed tochicken red blood cells (CRBC) (both Labor Dr. Merck & Kollegen,Ochsenhausen, Germany). For the HI assay, 50 μl of 2-fold dilutions ofpre-treated sera were incubated for 45 minutes with 4 hemagglutinationunits (HAU) of inactivated A/California/5 7/2009 (NIBSC, Potters Bar,UK) and 50 μl 0.5% CRBC were added.

Isolation of Bone Marrow and Memory Cell Analyses

Femurs and tibias were removed and both ends of the bone were cut withscissors. The marrow was flushed with RPMI-1640 (Lonza, Verviers,Belgium) using a 5m1 syringe (Norm-Ject, Tuttlingen, Germany) with 23Gneedle (Braun Medical, Emmenbrücke, Germany). Cluster cells weredissociated by vigorous pipetting. Red blood cells were removed using anRBC lysis buffer. Cells were counted and plated on the 96-well V bottomplate (3×10⁶ cells/well) for FACS staining. Cells were first incubatedfor 15 minutes at 4° C. with an anti-CD16/CD32 antibody (eBioscience,Frankfurt, Germany) to block unspecific binding followed by stainingwith PE-labelled HA-specific pentamer (H-2Kd IYSTVASSL, Proimmune,Oxford, UK) according to the manufacturer's instructions. Next, thecells were incubated with the following antibodies: CD44-FITC (1:200),Ly6C-PerCP-Cy5.5 (1:400), Thy1.2-APC (1:500), CD62L-PE-Cy7 (1:900),CD8α-APC-Cy7 (1:100) (BioLegend, Fell, Germany) and CD4-BD Horizon V450(1:900) (BD Biosciences). After 30 minutes incubation at 4° C. cellswere washed and stained with live/dead cell marker (AmCyan Aqua dye,Invitrogen, Life Technologies, Darmstadt, Germany) following by washingand FACS analyses using Fortessa or Canto II flow cytometer (BecktonDickinson, Heidelberg, Germany). Flow cytometry data were analysed usingFlowJo software (Tree Star, Inc., Ashland, Oreg., USA).

Intracellular Cytokine Staining

Splenocytes from vaccinated and control mice were isolated according toa standard protocol. Briefly, isolated spleens were grinded through acell strainer and washed in PBS/1% FBS followed by red blood cell lysis.After an extensive washing step with PBS/1% FBS splenocytes were seededinto 96-well plates (2×10⁶ cells/well). Cells were stimulated witheither Influenza Antigen A/California/7/2009 (5 μg/ml Health ProtectionAgency GB) or HAI (LYEKVKSQL) and HA2 (IYSTVASSL) peptides (5 μg/mleach, EMC Microcollections) and 2.5 μg/ml of an anti-CD28 antibody (BDBiosciences, Heidelberg, Germany) for 6 hours at 37° C. in the presenceof the mixture of GolgiPlug™/GolgiStop™ (Protein transport inhibitorscontaining Brefeldin A and Monensin, respectively; BD Biosciences).Cells incubated with medium or DMSO were used as controls, respectively.After stimulation cells were washed and stained for intracellularcytokines using the Cytofix/Cytoperm reagent (BD Biosciences Frankfurt,Germany) according to the manufacturer's instructions. The followingantibodies were used for staining: CD8-PECy7 (1:200), CD3-FITC (1:200),IL2-PerCP-Cy5.5 (1:100), TNFα-PE (1:100), IFNγ-APC (1:100)(eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) andincubated with Fey-block diluted 1:100. Aqua Dye was used to distinguishlive/dead cells (Invitrogen, Life Technologies, Darmstadt, Germany).Cells were collected using a Canto II flow cytometer (Beckton Dickinson,Heidelberg, Germany). Flow cytometry data were analysed using FlowJosoftware (Tree Star, Inc., Ashland, Oreg., USA).

Results

FIG. 3 shows that the intramuscular vaccination with a combination ofthe HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391)induces higher antibody titers against the HA protein compared tovaccination with the HA-mRNA (R2564) alone.

FIG. 4 shows that the intramuscular vaccination with a combination ofthe HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391)leads to significant increase in the number of central memory CD8+cells.

FIG. 5 shows that the intramuscular vaccination with a combination ofthe HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391)leads to significant increase in the number of central memory CD4+cells.

FIG. 6 shows that the intramuscular vaccination with a combination ofthe HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391)leads to significant increase in the number of multifunctional CD4+ Tcells.

Example 3: Induction of a Humoral and Cellular Immune Response AgainstHemagglutinin of Influenza Virus after Intramuscular Vaccination of MiceImmunization

On day zero, BALB/c mice were intramuscularly (i.m.) injected into bothM. tibialis with the influenza HA-encoding mRNA (R2564) alone or incombination the polymeric carrier cargo complex (RNA R2391) as shown inTable 3. Therein, the indicated amounts refer to the amount of RNA perse (see also Example 2 above). Mice injected with Ringer Lactate (RiLa)buffer served as controls. All animals received boost injections on day14. Blood samples were collected on days 14 and 21 for the determinationof serum anti-HA antibody titers in the hemagglutination inhibition(HAI) assay as in example 2. Spleens were harvested on day 21,splenocytes were isolated and T cells were analysed by intracellularcytokine staining as described in example 2.

TABLE 3 Animal groups Polymeric carrier HA cargo Vaccination Strain No.Route RiLa RNA complex schedule Group sex mice volume buffer R2564 R2391(day) 1 BALB/c 8 i.m. 2 × 25 — — d0: prime, Female 2 × 25 μl d14: boostμl 2 BALB/c 8 i.m. — 2 × 20 — d0: prime, Female 2 × 25 μg d14: boost μl3 BALB/c 8 i.m. — 2 × 20 2 × 20 μg d0: prime, Female 2 × 25 μg d14:boost μl

Results

As can be seen in FIG. 7, the intramuscular vaccination with 40 μgHA-mRNA (R2564) combined with 40 μg of polymeric carrier cargo complex(R2391) induced elevated numbers of IFNγ/TNFα double-positivemultifunctional CD4+ T cells as determined by intracellular cytokinestaining after stimulation with Influenza Antigen A/California/7/2009compared to vaccination with 40 μg of HA-mRNA (R2564) alone.

Example 4: Induction of a Humoral Immune Response Against Hemagglutininof Influenza Virus H1N1 after Intramuscular Vaccination of Pigs

Domestic pigs were screened for swine influenza using the hemagglutinininhibition assay at the breeding facility. Only seronegative pigs wereintroduced into the study.

Animal Groups and Treatment:

Vaccination schedule Group Animals No. Left leg i.m. (day) 1 Femaledomestic pig, 5 200 μg R2630 d 1: prime, Hungarian large whiteRNActive ® d 29: boost 2 Female domestic pig, 5 200 μg d 1: prime,Hungarian large white R2564 + d 29: boost 200 μg R2391 3 Female domesticpig, 5 200 μg d 1: prime, Hungarian large white R2564pA d 29: boost 4Female domestic pig, 5 200 μg d 1: prime, Hungarian large whiteR2564pA + d 29: boost 200 μgR2391

The RNA formulations prepared according to Example 1 were injectedintramuscularly into the left hind leg. The treatment days were studyday 1 and 29. Blood samples were taken on day −7, day 29,day 43 and day57.

Hemagglutination Inhibition Assay:

For the hemagglutination inhibition (HI) assay, pig sera were treatedwith RDE (II) “SEIKEN” (WAK-Chemie Medical GmbH, Steinbach/Ts, Germany)o/n at 37° C., heat inactivated (56° C., 60 min), incubated with kaolin(Labor Dr. Merck & Kollegen, Ochsenhausen, Germany), and pre-adsorbed tochicken red blood cells (CRBC) (Lohmann Tierzucht, Cuxhaven, Germany).For the HI assay, 50 μl of 2-fold dilutions of pre-treated sera wereincubated for 45 minutes with 4 hemagglutination units (HAU) ofinactivated A/California/5 7/2009 (NIBSC, Potters Bar, UK) and 50 μl0.5% CRBC were added.

Results

As can be seen in FIG. 8, the intramuscular vaccination with 200 μg ofHA-mRNA (R2564) combined with 200 μg of polymeric carrier cargo complex(R2391) induced elevated neutralizing antibody titers against the HAprotein compared to vaccination with the HA-mRNA vaccine (R2630RNActive®) without the polymeric carrier cargo complex (R2391).Enzymatic polyadenylation increased the neutralizing antibody titersinduced by HA-encoded mRNA (R2564pA), but also in this case the additionof the polymeric carrier cargo complex (R2391) further increased theneutralizing antibody titers against the HA protein.

Example 5: Induction of Virus Neutralization Titers Against Rabies Virusafter Intramuscular Vaccination of Mice

Balb/c mice were vaccinated 2 times (d0 and d21) with 20 μg RAV-G mRNA(R2506) or enzymatically polyadenylated RAV-G mRNA (R3344) alone or incombination with 40 μg RNAdjuvant prepared according to Example 1 intoboth M. tibialis. Therefore, 8 animals (group A) were vaccinated i.m.with R2506 (naked RAV-G mRNA), 8 animals (group B) were vaccinated i.m.with R3344 (enzymatically polyadenylated R2506—naked RNA), 8 animals(group C) were vaccinated i.m. with R2506 (naked RAV-G mRNA) incombination with RNAdjuvant and 8 animals (group D) were vaccinated i.m.with R3344 (enzymatically polyadenylated naked RAV-G mRNA) incombination with RNAdjuvant®. 8 mice injected with Ringer-Lactatesolution (RiLa) served as negative controls. Blood was collected 28 daysafter prime (7 days after boost). Serum was analyzed for virusneutralization titers (VNT).

Animal Groups and Treatment

group n mice RNA RNAdjuvant vaccination A 8 Balb/c 20 μg R2506 — d 0, d21 B 8 Balb/c 20 μg R3344 — d 0, d 21 C 8 Balb/c 20 μg R2506 40 μg R2391d 0, d 21 D 8 Balb/c 20 pg R3344 40 μg R2391 d 0, d 21 E 8 Balb/c RiLa —d 0, d 21

Virus Neutralization Test

The virus neutralizing antibody response (specific B-cell immuneresponse) was detected by using a virus neutralisation assay. The resultof that assay is referred to as virus neutralization titer (VNT).According to WHO standards, an antibody titer is considered protectiveif the respective VNT is at least 0.5 IU/ml. Therefore, blood sampleswere taken from vaccinated mice on day 28 and sera were prepared. Thesesera were used in fluorescent antibody virus neutralisation (FAVN) testusing the cell culture adapted challenge virus strain (CVS) of rabiesvirus as recommended by the OIE (World Organisation for Animal Health)and first described in Cliquet F., Aubert M. & Sagne L. (1998); J.Immunol. Methods, 212, 79-87. Shortly, heat inactivated sera are testedin microplates as quadruplicates in serial two-fold dilutions for theirpotential to neutralize 100 TCID₅₀ (tissue culture infectious doses 50%)of CVS in a volume of 50 μl. Therefore, sera dilutions were incubatedwith virus for 1 hour at 37° C. (in humid incubator with 5% CO₂) andsubsequently trypsinized BHK-21 cells were added (4×10⁵ cells/ml; 50 μlper well After an incubation period of 48 hours in humid incubator at37° C. and 5% CO₂, cells were fixed in 80% aceton at room temperaturefor 30 minutes. Infection of cells was analysed using FITC anti-rabiesconjugate (30 minutes at 37° C.). Plates were washed twice using PBS andexcess of PBS was removed. Cell cultures are scored positive or negativefor the presence of rabies virus. For each well, the presence or absenceof fluorescent cells is evaluated. Wells with no detectable fluorescentcell are scored negative. Negative scored sera treated wells representneutralization of rabies virus. Each FAVN tests includes WHO or OIEstandard serum (positive reference serum) that serves as reference forstandardisation of the assay. Neutralization activity of test sera wascalculated with reference to the standard serum provided by the WHO anddisplayed as International Units/ml (IU/ml).

Results

As can be seen in FIG. 9, the intramuscular vaccination with 20 μg ofnaked RAV-G mRNA (R2506) or enzymatically polyadenylated naked RAV-GmRNA (R3344) combined with 40 μg of polymeric carrier cargo complex(R2391; RNAdjuvant) induced elevated virus neutralization titerscompared to vaccination with the RAV-G mRNAs alone.

Example 6: Reduction of RSV Virus Titers in the Lung after Vaccinationwith mRNA Encoding RSV F Protein Groups and Treatment:

Treatment Route, Immunisation Group Strain/sex Nr. RNA/mouse Volumeschedule challenge A Cotton rats/ 5 R2682 i.m. d0, d14 d49 female 80 μg1 × 100 μl B Cotton rats/ 5 R2391 i.m. d0, d14 d49 female 40 μg + 1 ×100 R2682 μl 40 μg C Cotton rats/ 5 RiLa i.d. d0, d14 d49 female 2 × 50D Cotton rats/ 5 Live μl d0 d49 female RSV/A2 E Cotton rats/ 5 untreated— — d49 female

Cotton rats represent an established and widely accepted animal modelfor RSV that is routinely used for the development of RSV vaccines.Cotton rats respond to formalin-inactivated RSV virus vaccinepreparations with enhanced lung pathology. This allows the evaluation ofthe safety of a vaccination in terms of enhanced disease phenomenon.

In order to assess the effect of the RSV-F encoding mRNA (R2682), themRNA was administered intramuscularly on day 0 and 14 either alone or incombination with the polymeric cargo complex (R2391; RNAdjuvant) asshown above. An additional group was immunized intramuscularly (i.m.)with live RSV/A2 (Sigmovir) (10⁵ plaque forming units, pfu) to comparetheir immunogenicity to mRNA vaccines. After immunization, the cottonrats were challenged by intranasal (i.n.) infection with RSV/A2 virus(105 PFU in 100 μl; Sigmovir). On day 54 the lung was harvested en blocfor viral titration.

Results:

As shown in FIG. 10, intramuscular vaccination with 40 μg of naked RSV-FmRNA (R2682) combined with 40 μg of polymeric carrier cargo complex(R2391; RNAdjuvant) led to significantly reduced viral titers in thelung compared to vaccination with the RSV-F mRNA alone.

1. A polymeric carrier cargo complex, comprising: a) as a carrier apolymeric carrier formed by disulfide-crosslinked cationic components,and b) as a cargo at least one first nucleic acid molecule, for use asan immunostimulating agent or as an adjuvant, wherein the polymericcarrier cargo complex is administered in combination with at least onesecond nucleic acid molecule encoding a protein or a peptide, andwherein the polymeric carrier cargo complex and the second nucleic acidmolecule are administered intramuscularly.
 2. A polymeric carrier cargocomplex, comprising: a) as a carrier a polymeric carrier formed bydisulfide-crosslinked cationic components, and b) as a cargo at leastone first nucleic acid molecule, for use as an immunostimulating agentor as an adjuvant, wherein the polymeric carrier cargo complex isadministered in combination with at least one second nucleic acidmolecule encoding a protein or a peptide, wherein the second nucleicacid molecule is an RNA molecule.
 3. The polymeric carrier cargo complexfor use as an immunostimulating agent or an adjuvant according to claim1, wherein the second nucleic acid molecule is an RNA molecule.
 4. Thepolymeric carrier cargo complex for use as an immunostimulating agent oran adjuvant according to any one of claims 1 to 3, wherein the secondnucleic acid molecule is either naked or complexed with a cationiccomponent.
 5. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 1to 4, wherein the second nucleic acid molecule is not packaged in aparticle, such as a virus particle, an inactivated virus particle or avirus-like particle.
 6. The polymeric carrier cargo complex for use asan immunostimulating agent or an adjuvant according to any one of claims1 to 4, wherein the second nucleic acid molecule is not comprised in thepolymeric carrier cargo complex.
 7. The polymeric carrier cargo complexfor use as an immunostimulating agent or an adjuvant according to anyone of claims 1 to 6, wherein the polymeric carrier cargo complex andthe second nucleic acid molecule are not administered together with aprotein or peptide antigen selected from the group consisting of anantigen from a pathogen associated with infectious disease, an antigenassociated with allergy or allergic disease, an antigen associated withautoimmune disease, an antigen associated with a cancer or tumourdisease, or a fragment, variant and/or derivative of said protein orpeptide antigen.
 8. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 1to 6, wherein the polymeric carrier cargo complex and the second nucleicacid molecule are not administered together with a protein or peptideantigen.
 9. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 1to 8, wherein the second nucleic acid molecule is an mRNA molecule. 10.The polymeric carrier cargo complex for use as an immunostimulatingagent or an adjuvant according to claim 9, wherein the mRNA moleculecomprises at least one selected from the group consisting of a 5′-UTR, a3′-UTR, a poly(A) sequence, a poly(C) sequence and a histone stem-loopsequence.
 11. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to claim 10, whereinthe 3′-UTR comprises a nucleic acid sequence derived from the 3′-UTR ofan albumin gene.
 12. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to claim 10 or 11,wherein the 3′-UTR comprises the nucleic acid sequence corresponding toSEQ ID NO.
 388. 13. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 10to 12, wherein the histone stem-loop sequence comprises a nucleic acidsequence corresponding to SEQ ID NO.
 389. 14. The polymeric carriercargo complex for use as an immunostimulating agent or an adjuvantaccording to any one of claims 10 to 13, wherein the 5′-UTR comprises anucleic acid sequence derived from a 5′-UTR of a TOP gene.
 15. Thepolymeric carrier cargo complex for use as an immunostimulating agent oran adjuvant according to any one of claims 10 to 14, wherein the 5′-UTRcomprises a nucleic acid sequence derived from a ribosomal protein gene.16. The polymeric carrier cargo complex for use as an immunostimulatingagent or an adjuvant according to any one of claims 10 to 15, whereinthe 5′-UTR comprises a nucleic acid sequence derived from ribosomalprotein 32L gene.
 17. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 9to 16, wherein the mRNA molecule comprises a nucleic acid sequencederived from a 5′-TOP-UTR, a GC-optimized coding sequence, a nucleicacid sequence derived from the 3′-UTR of an albumin gene, apoly(A)-sequence, a poly(C)-sequence, and a histone stem loop.
 18. Thepolymeric carrier cargo complex for use as an immunostimulating agent oran adjuvant according to any one of claims 1 to 17, wherein the at leastone first nucleic acid molecule is an RNA molecule.
 19. The polymericcarrier cargo complex for use as an immunostimulating agent or anadjuvant according to any one of claims 1 to 18, wherein the at leastone first nucleic acid molecule is an immunostimulatory nucleic acid,preferably a non-coding immunostimulatory nucleic acid.
 20. A polymericcarrier cargo complex for use as an immunostimulating agent or anadjuvant according to any one of claims 1 to 19, wherein thenitrogen/phosphate (N/P) ratio of the cationic components to the atleast one first nucleic acid molecule is in the range of 0.1-20, or inthe range of 0.1-5, or in the range of 0.1-1.
 21. A polymeric carriercargo complex for use as an immunostimulating agent or an adjuvantaccording to any one of claims 1 to 20, wherein the polymeric carriercomprises functional peptides or proteins additionally to the cationiccomponents.
 22. A polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to claim 21, whereinthe functional peptides or proteins are peptide or protein antigens orantigen epitopes.
 23. A polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 1to 22, wherein the polymeric carrier additionally comprises a ligand.24. A polymeric carrier cargo complex for use as an immunostimulatingagent or an adjuvant according to claim 23, wherein the ligand ismannose.
 25. The polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 1to 24, wherein the cationic components are cationic peptides, preferablyselected from oligocationic and polycationic peptides.
 26. The polymericcarrier cargo complex for use as an immunostimulating agent or anadjuvant according to claim 25, wherein the cationic peptides areselected from peptides according to formula (I)(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+m+n+o+x=3-100, and l, m, n or o=independently of each other is anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90and 91-100, provided that the overall content of Arg, Lys, His and Ornrepresents at least 10% of all amino acids of the cationic peptide; andXaa is any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x=any numberselected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90,provided, that the overall content of Xaa does not exceed 90% of allamino acids of the cationic peptide, or are selected from peptidesaccording to subformula (Ia){(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)} or frompeptides according to subformula (Ib)Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂ wherein(Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o); and x are as defined above;Xaa′ is any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His, Orn; or Cys and y is anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and81-90, provided that the overall content of Arg (Arginine), Lys(Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% ofall amino acids of the oligopeptide and wherein Cys₁ and Cys₂ areCysteines proximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x).
 27. The polymericcarrier cargo complex for use as an immunostimulating agent or anadjuvant according to claim 25 or 26, wherein the disulfide-bonds areformed by cysteine residues contained in the cationic peptides.
 28. Apolymeric carrier cargo complex for use as an immunostimulating agent oran adjuvant according to claim 27, wherein the cysteine residue islocated proximal to, preferably at the terminal ends of the cationicpeptides.
 29. A polymeric carrier cargo complex for use as animmunostimulating agent or an adjuvant according to any one of claims 1to 28, wherein the cationic component comprises the peptide CysArg₁₂Cys.30. A polymeric carrier cargo complex for use as an immunostimulatingagent or an adjuvant according to any one of claims 1 to 29 comprising:a. as a carrier a polymeric carrier formed by disulfide-crosslinkedcationic polymers, and b. as a cargo at least one nucleic acid(molecule), in the treatment or prophylaxis of a disease selected from atumour or a cancer disease, an infectious disease, an autoimmune diseaseor an allergy.
 31. A pharmaceutical composition comprising: (A) apolymeric carrier cargo complex, comprising: a) as a carrier a polymericcarrier formed by disulfide-crosslinked cationic components, and b) as acargo at least one first nucleic acid molecule, and (B) at least onesecond nucleic acid molecule, wherein the at least one second nucleicacid molecule is an RNA molecule encoding a protein or a peptide. 32.The pharmaceutical composition according to claim 31, wherein the atleast one second nucleic acid molecule encodes a protein or peptideantigen that is selected from the group consisting of an antigen from apathogen associated with infectious disease; an antigen associated withallergy; an antigen associated with autoimmune disease; and an antigenassociated with cancer or tumour disease; or a fragment, variant and/orderivative of said antigen.
 33. The pharmaceutical composition accordingto claim 31 or 32, wherein component (B) is not covalently linked to(A).
 34. The pharmaceutical composition according to any one of claims31 to 33, which does not comprise a protein or peptide antigen selectedfrom the group consisting of an antigen from a pathogen associated withinfectious disease, an antigen associated with allergy or allergicdisease, an antigen associated with autoimmune disease, an antigenassociated with a cancer or tumour disease, or a fragment, variantand/or derivative of said protein or peptide antigen.
 35. Thepharmaceutical composition according to any one of claims 31 to 33,which does not comprise a protein or peptide antigen.
 36. Thepharmaceutical composition according to any one of claims 31 to 35,wherein the second nucleic acid molecule is either naked or complexedwith a cationic component.
 37. The pharmaceutical composition accordingto any one of claims 31 to 36, wherein the the second nucleic acidmolecule is not packaged in a particle, such as a virus particle, aninactivated virus particle or a virus-like particle.
 38. Thepharmaceutical composition according to any one of claims 31 to 37,wherein the antigen is derived from a pathogen, preferably a viral,bacterial, fungal or protozoan pathogen, preferably selected from thelist consisting of: Rabies virus, Hepatitis B virus, human Papillomavirus (hPV), Bacillus anthracis, Respiratory syncytial virus (RSV),Herpes simplex virus (HSV), Dengue virus, Rotavirus, Influenza virus andMycobacterium tuberculosis.
 39. The pharmaceutical composition accordingto any one of claims 31 to 37, wherein the antigen is associated withallergy or allergic disease and is derived from a source selected fromthe list consisting of: grass pollen, tree pollen, flower pollen, herbpollen, dust mite, mold, animals, food, and insect venom.
 40. Thepharmaceutical composition according to any one of claims 31 to 37,wherein the antigen is associated with a cancer or tumour disease and isselected from the list consisting of: p53, CA125, EGFR, Her2/neu, hTERT,PAP, MAGE-A1, MAGE-A3, MAGE-C1, MAGE-C2, Mesothelin, MUC-1, NY-ESO-1,GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA, VEGF, VEGFR1, VEGFR2, Ras,CEA, Survivin, 5T4, STEAP and WT1.
 41. The pharmaceutical compositionaccording to any one of claims 31 to 40, wherein the polymeric carriercargo complex is for use as an immunostimulating agent or as anadjuvant.
 42. The pharmaceutical composition according to any one ofclaims 31 to 41, wherein the at least one first nucleic acid molecule isan RNA.
 43. The pharmaceutical composition according to any one ofclaims 31 to 42, wherein the at least one first nucleic acid molecule isan immunostimulatory nucleic acid.
 44. The pharmaceutical compositionaccording to any one of claims 31 to 43, wherein the nitrogen/phosphate(N/P) ratio of the cationic components to the at least one first nucleicacid molecule is in the range of 0.1-20, or in the range of 0.1-5, or inthe range of 0.1-1.
 45. The pharmaceutical composition according to anyone of claims 31 to 44, wherein the polymeric carrier additionallycomprises a ligand.
 46. The pharmaceutical composition according toclaim 45, wherein the ligand is mannose.
 47. The pharmaceuticalcomposition according to any one of claims 31 to 46, wherein thecationic components are cationic peptides.
 48. The pharmaceuticalcomposition according to any one of claims 31 to 47, wherein thecationic peptides are selected from peptides according to formula (I)(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+M+n+0+x=3-100, and l, m, n or o=independently of each other is anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90and 91-100, provided that the overall content of Arg, Lys, His and Ornrepresents at least 10% of all amino acids of the cationic peptide; andXaa is any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x=any numberselected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90,provided, that the overall content of Xaa does not exceed 90% of allamino acids of the cationic peptide, or are selected from peptidesaccording to subformula (Ia){(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)} or frompeptides according to subformula (Ib)Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂ wherein(Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o); and x are as defined above;Xaa′ is any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His, Orn; or Cys and y is anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and81-90, provided that the overall content of Arg (Arginine), Lys(Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% ofall amino acids of the oligopeptide and wherein Cys₁ and Cys₂ areCysteines proximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x).
 49. Thepharmaceutical composition according to claim 47 or 48, wherein thedisulfide-bonds are formed by cysteine residues contained in thecationic peptides.
 50. The pharmaceutical composition according to claim49, wherein the cysteine residue is located proximal to, preferably atthe terminal ends of the cationic peptides.
 51. The pharmaceuticalcomposition according to claim 49 or 50, wherein the cationic componentcomprises the peptide CysArg₁₂Cys.
 52. A vaccine, comprising apharmaceutical composition according to any one of claims 31 to
 51. 53.The vaccine according to claim 52, wherein the pharmaceuticalcomposition according to any of claims 31 to 51 elicits an adaptiveimmune response.
 54. The vaccine according to claim 52 or 53, whereinthe polymeric carrier cargo complex is used as an immunostimulatingagent or adjuvant.
 55. The vaccine according to any one of claims 52 to54, wherein the vaccine further comprises a pharmaceutically acceptablecarrier.
 56. A kit, preferably a kit of parts, comprising thepharmaceutical composition according to any one of claims 31 to 51,and/or the vaccine according to any one of claims 52 to 55, andoptionally a liquid vehicle for solubilising and optionally technicalinstructions with information on the administration and dosage of theactive composition and/or the vaccine.
 57. The kit according to claim56, wherein the at least one second nucleic acid molecule, which encodesa protein or peptide, is provided in lyophilized form as a separatepart.
 58. The kit according to claim 56 or 57, wherein the kit containsas a part Ringer-Lactate solution.
 59. The pharmaceutical compositionaccording to any one of claims 31 to 51 or the kit according to any oneof claims 56 to 58, for use in the treatment or prophylaxis of aninfectious disease; an allergy; an autoimmune disease; or a cancer ortumour disease.